Methods and systems for printing biological material

ABSTRACT

The present disclosure provides methods and systems for printing a three-dimensional (3D) material. In some examples, a method for printing a 3D biological material comprises providing a media chamber comprising a medium comprising (i) a plurality of cells and (ii) one or more polymer precursors. Next, at least one energy beam may be directed to the medium in the media chamber along at least one energy beam path that is patterned into a 3D projection wherein the x, y, and z dimensions may be simultaneously accessed in accordance with computer instructions for printing the 3D biological material in computer memory, to form at least a portion of the 3D biological material comprising (i) at least a subset of the plurality of cells, and (ii) a polymer formed from the one or more polymer precursors.

CROSS-REFERENCE

This application is a continuation of International Application No.PCT/US2018/021850, filed on Mar. 9, 2018, which claims the benefit ofU.S. Provisional Patent Application No. 62/469,948, filed Mar. 10, 2017,both of which are incorporated herein by reference in their entireties.

BACKGROUND

Despite significant advances in the fields of cell biology,microfluidics, engineering, and three-dimensional printing, to date,conventional approaches have failed to re-create functional capillariesthat feed and support the thick tissue necessary to construct a humanorgan. To date, these approaches in tissue engineering have relied onthe in-growth of blood vessels into tissue-engineered devices to achievepermanent vascularization. This strategy has worked for some tissuesthat are either very thin such as a bladder wall replacement or tissuessuch as bone replacements that do not require vasculature to function.However, current tissue engineering techniques fall short in thecreation of complex tissues such as large vital organs, including liver,kidney, thick skin, and heart. Larger tissues can also be thought of asan organization of smaller tissue sub-units; for example, the kidney iscomprised of hundreds of thousands of nephron units, the functional unitof the lungs, i.e., the alveolar spaces, have a combined surface area of70 to 80 meters squared (m²), but are only 1 cell wall, 5 to 10micrometers (μm), thick. Current tissue printing methodology not onlyfails to re-create the fine microvasculature necessary to supporttissues thicker than 300 micrometers (μm), but cannot organize cellsinto the structural orientations and niches that are necessary for organfunction.

Multi-photon laser based excitation is used in chemistry and physics forthe generation of microstructures at sub-nanometer resolutions usingphoto-polymerization reactions, where photopolymerization is thelight-based polymerization of a material. The use of two-photonmicroscopy to induce polymerization was first described in 1981. It wassubsequently used to construct micrometer-to-nanometer-scale parts andtools by raster-scanning the pin-point focused laser in the x-ydimensions, tracing out the structure line by line. The high-resolutionpin-point of the two-photon excitation allowed for sub-nanometer printresolution and additive manufacturing with plastics that may bephoto-polymerized. As two-photon technology evolved and two-photonexcitation was combined with laser-scanning microscopy, imaging ofliving tissues became possible. Long-wavelengths, typically greater than700 nanometers (nm) used in multi-photon excitation allowed for greatertissue penetration due to reduced Rayleigh scattering, minimalphoto-bleaching of fluorescent probes, and minimal to no detectabletissue toxicity. Thus, multi-photon laser excitation became useful inbiology for non-invasive imaging of tissues at depths greater than thoseachievable with single-photon laser based confocal imaging.

Extended time periods of live-cell imaging, typically greater than a fewhours, through the use of endogenous probes that are excited by two- andthree-photon absorption were described shortly thereafter. One of thefirst applications of two-photon imaging was in the field of botany,which first described the inherently low toxicity of two-photonexcitation to living cells. With its use in the study of neuronalsignaling, two-photon microscopy was demonstrated to be an importantlow-toxicity photo-imaging tool in mammalian cells, wherein two-photonexcitation was mild enough to not trigger the firing of a single neuronuntil external stimulus was applied. In 2002, video-rate two-photonimaging was demonstrated to be non-toxic to mammalian living cells inwhole tissues, such as lymph nodes. Later, living cells in skin, lung,spleen, liver, and various other tissues were examined using two-photonmicroscopy. Extended imaging time courses are now up to 24 hours withoutobservable negative effects on the biological activity or viability ofcells, and have been conducted in a number of mammalian cells andtissues.

Together, high viability of individual cells combined with the abilityto polymerize biomaterials have made multi-photon excitation an idealtool for polymerization of materials in both cell-free medium and mediumthat contain cells to be embedded. Thus, multi-photon laser basedexcitation has been used for tissue engineering of somethree-dimensional tissue structures, however significant limitations inthe speed associated with printing complex structures with 2-dimensionalraster scanning as described with current technology make the creationof complex, multicellular three-dimensional tissue structures includingfunctional organs to date, infeasible. This is due to the inherent tradeoff in manufacturing speed and resolution for a single-unit (scan line)process. For example, it is estimated that raster-scanning of a laser ata resolution fine enough to create a centimeter cubed of tissue may takeover three decades to perform. However, in the field of tissueengineering, the prospect of three-dimensional tissue structures offersa hope of promising treatment options to patients in need of organtransplantation and improved drug discovery and investigation based onwhole, human-derived tissues rather than animal models, which aresubject to error.

Multi-photon excitation methods of tissue engineering have been shown tobe superior to extrusion or spray-based printing techniques which relyupon deposition of materials forced through a nozzle into a predefinedpattern or form generating structure. Spray or droplet based cellprinting lacks resolution, biocompatibility, is relatively slow, and isnot scalable such that whole tissues cannot be built. Microvasculatureor capillaries that are present in all organs and tissues are a maximumof e.g. 5 to 10 micrometers in diameter. This finely structuredvasculature is necessary for tissue to be viable. Current droplet orspray printing methods do not have the capability of producing vascularstructures smaller than 50 micrometers. Therefore, to date, no tissuestructure thicker than roughly e.g. 250 to 300 micrometers, which is thelimit of oxygen diffusion and waste exchange, has been reported to beviable. Without proper perfusion from microvasculature, tissues thickerthan e.g. 250 to 300 micrometers become hypoxic and starve fornutrients, eventually becoming necrotic and dying from the inside out.Furthermore, single-cell layers and fine branching necessary to printthe microvasculature necessary for support of tissue integrity andfunction cannot be achieved using current extrusion or droplet-basedprinting methods.

Lack of resolution at the cellular level further limits the developmentof complex or small scale three dimensional structures and cell nichesthat are dependent on direct interactions of multiple cell types orlayers of cells. Pre-printing of fine structures or using pre-printedacellular scaffolds can achieve the resolution necessary to createmicrovasculature, however these structures require cell seeding,limiting the ability to place cells in specific orientations or niches,increasing development time of tissues, and leading to reduced cellviability as many cell seeding techniques require force to embed cellsinto small porous structures. Furthermore, the rigidity of printedscaffold structures impedes development of fully compatible biologicallyfunctional tissues by limiting cell-guided architectural and structuralchanges and by limiting the development of new blood vessels.Additionally, the rigidity of printed scaffold structures limitsfunctional niche-adapting architectural changes, which preventsvascularized tissue development since cells are fixed and only a fewtypes of cells can be deposited in such rigid structures.

In extrusion printing, cell viability is also compromised and print timeis too slow to maintain cell viability over an extended period of timenecessary to print a tissue structure. The scalability of extrusionprinting is also inherently limited such that it does not solve issuesof resolution and thus, extrusion printing does not have the capabilityto print tissues at a large scale. The size and functionality of printedtissues is limited by a series of factors. These limiting factorsinclude the inability to scale to a larger organ sized structure (timeto print), cell viability during the printing process, ability to createmulticellular three dimensional niches, inflexibility of the finalstructure such that natural cell-induced development can occur, and thelack of microvasculature that only allows the maximum tissue thicknessto be e.g. 250 to 300 micrometers. Thus, the utility of currentextrusion printing techniques in tissue engineering is limited by theextensive time to production and the lack of fine enough resolution toaccurately produce complex, vascularized three dimensional structures.Additionally, extrusion techniques often do not allow for cells to beplaced directly in the print medium, are not biocompatible, and do notallow for changes in structure through the developmental process.

Two-photon excitation has been employed in the field of tissuemicrofabrication to speed time to production and improve printingresolution. Pulsed, two-photon lasers are wavelength-tunable and producepinpoint, ultrafast, subnanometer-resolution polymerization ofmaterials. Two-photon photo-polymerization has been applied to isolationof single cell types within defined structures. However, the technologyfor two-photon encapsulation of cell technology is significantly limitedfor multiple reasons, primarily the time associated with step-wiseadditive creation of a three-dimensional structure and therefore cannotyield the necessary complex capillary networks or multicellularstructures necessary to produce vascularized tissues.

Primary limitations include the issue that these techniques are notalways biocompatible, in large part due to reliance on biologicallyincompatible photo-initiators. Further limitations include, but are notlimited to the speed of printing such that only small structurescontaining only a few cells are capable of being produced within thewindow of cell viability. Therefore, current two-photon cellencapsulation methods cannot generate large enough tissue structures tobe useful for organ transplant and for most tissue-based applications.In addition, there are no provisions that allow for the necessary flowof cell supra-structures during tissue growth and development, and forthe promotion of cell-cell contact, both of which are necessary for thegrowth and development of functional tissues. There are no provisionsfor additive cell layers to be introduced. And, finally, there are noprovisions for specific structuring of networks that allow for certainforces of tension to be applied to a bed of developing cells, anecessary step in vascular development.

Current printing or deposition technologies do not allow for specificcell movement, cell-cell interactions, and the resultant development oftissue structures. These cell-intrinsic behaviors that occur naturallyduring development are essential for the formation of viable tissues.Tissues are three-dimensional structures comprised of cells in variousstates of differentiation, each with a specific function. Development ofa biologically-active and self-maintaining tissue that can effectivelyperform as a replacement tissue requires proper localization of cellsrelative both to other cells and to such structural elements asvasculature, tendon, bone matrices, and other structural components oftissue. Differentiation is often driven by genetic changes, which maydirectly result in structural changes, such as involution, significantshifts in structure and/or may facilitate tissue development throughchanges in expression of tissue-specific structural, functional, orsignaling proteins. During morphogenesis, wound repair, and cancerinvasion, for example, cells move collectively as large sheets, strands,or clusters allowing for rapid contact-based force generation orcollective polarization. These cell movements en masse are necessary forproper tissue formation and function. These cell movements and exposureto different environmental forces, such as blood flow, are causative ofcritical developmental cues for differentiation of functional tissues.For example, as arterial and venous capillaries are formed from uniformprecursor cells that undergo significant shifts in gene expression thatdrives phenotype differentiation and results in functional vascularstructures arising from the same cell type. Furthermore, support for andallowance of structural tolerance of stretch and pressure changes isnecessary for vascular function and maintenance of vascular endothelialcell identity. Indeed, vascular walls have elastic fiber deposition asone of their primary components. Despite the numerous methods to developblood vessels with bioprinting techniques, no current process reportedor hypothesized allows for the combined deposition of strand-basedstructures for creation of cell niches for vascular development or cellmovement in sheet or strand components. In short, currently developedand described structures for tissue based cell printing are not designedto tolerate or support these forces, and proper vascularization ormicrovascular (capillary) development has yet to be demonstrated.Without vascularization with capillaries, printed cells can only survivein extremely thin sheets that are limited to, e.g., 200-300 micrometersby oxygen and nutrient diffusion to provide for waste and nutrientexchange. Therefore, it may not be possible to develop a functionaltransplantable tissue without microvasculature.

SUMMARY

Recognized herein are various issues with previously described methodsfor printing of cells into tissue-forming structures. Such methods maybe limited by (i) the sheer variety of cells necessary to create acomplex vascularized tissue; (ii) rigid structures that do not allow forthe supra-structural movement that occurs as a necessary element of, orfacilitates developmental changes necessary for further differentiation;(iii) elements of varied structural rigidity or moment that canfacilitate or adapt to cell-cell interactions between like or unlikecell layers during development, (iv) lack of cell-type specific channelsor nets; and (v) lack of pre-printed (first step) or reprinted(intermediate or final step) vascular cells, (vi) structures designed toallow for cell-cell interactions while withstanding mechanical forcesincluding pressure, tension, twisting, stretch, or motion necessary forvascular development, differentiation, and function, within tissues.

Additionally, single photon raster-scan printing and two dimensionalprojection of a sheet of light may be both significantly slower asmanufacturing processes than the methods and systems provided herein. Insome instances, it may be estimated that a structure that would takedecades to create with single-photon raster scanning, and weeks in thecase of 2D projection, may be created in a matter of 24 hours or fewerwith the three-dimensional (3D) holographic printing methods and systemsprovided herein. Furthermore, the 3D holographic printing methods andsystems provided herein may be fill-factor independent when the entirestructure is projected at once because the printing occurssimultaneously at all points within a cubic volume. Therefore, aprinting speed may be decoupled from a resolution when using aholographic printing projection such as the one used by the 3Dholographic printing methods and systems provided herein. A printingspeed may be volume dependent, and the print volume may be dictated bystatic optical components, when using the 3D holographic printingmethods and systems provided herein.

Thus far, the field of tissue engineering may have failed to produceresponsive, biologically-active vascularized tissues that behave asnative tissue in large enough structures be useful for transplantation,systems-integrative drug testing, and/or development of biologictherapeutics, such as human antibodies.

Two-photon lasers may provide a low cellular toxicity profile basedon: 1) pinpoint sites of two-photon excitation that fall off as afunction of the square of the distance from the focal point in the x, y,and z dimensions such that peak laser power may not be spread throughoutthe sample; 2) rapid x, y scanning of the excitation point minimizingthe time a cell may be exposed to the peak laser power at the point ofexcitation; and 3) ultra-short, sub-picosecond pulse widths that mayallow for gaps in time where few to no photons are engaging the materialor cells.

In an aspect, the present disclosure provides a method for printing athree-dimensional (3D) biological material, comprising: (a) providing amedia chamber comprising a medium comprising (i) a plurality of cellsand (ii) one or more polymer precursors; and (b) directing at least oneenergy beam to the medium in the media chamber along at least one energybeam path that is patterned into a 3D projection in accordance withcomputer instructions for printing the 3D biological material incomputer memory, to form at least a portion of the 3D biologicalmaterial comprising (i) at least a subset of the plurality of cells, and(ii) a polymer formed from the one or more polymer precursors.

In some embodiments, the biological material develops into abiologically functional tissue. In some embodiments, the method furthercomprises prior to (b), generating a point-cloud representation orlines-based representation of the 3D biological material in computermemory, and using the point-cloud representation or lines-basedrepresentation to generate the computer instructions. In someembodiments, the method further comprises converting the point-cloudrepresentation or lines-based representation into an image.

In some embodiments, the image is projected in a holographic manner. Insome embodiments, the image is deconstructed and reconstructed prior toprojection in a holographic manner. In some embodiments, the point-cloudrepresentation or the lines-based representation comprisesmulti-dimensional structural elements corresponding to the 3D biologicalmaterial. In some embodiments, the point-cloud representation or thelines-based representation comprises structural elements in twodimensions, wherein the structural elements are associated with tissuefunction and/or cellular segregation. In some embodiments, point-cloudrepresentation or the lines-based representation comprises structuralelements in three dimensions, wherein the structural elements areassociated with tissue function and/or cellular segregation.

In some embodiments, the at least one energy beam comprises coherentlight. In some embodiments, the at least one energy beam is generated bya laser. In some embodiments, the at least one energy beam is phasemodulated. In some embodiments, the one or more polymer precursorscomprise at least two different polymeric precursors.

In some embodiments, the method further comprises repeating (b) alongone or more additional energy beam paths to form at least anotherportion of the 3D biological material. In some embodiments, the at leastanother portion of the 3D biological material is linked to the 3Dbiological material formed in (b). In some embodiments, the at leastanother portion of the 3D biological material is not linked to the 3Dbiological material formed in (b). In some embodiments, (b) furthercomprises directing at least two energy beams to the medium in the mediachamber along at least two energy beam paths in accordance with thecomputer instructions, to permit multiple portions of the medium in themedia chamber to simultaneously form at least a portion of the 3Dbiological material.

In some embodiments, the at least two energy beams are of identicalwavelengths. In some embodiments, the at least two energy beams are ofdifferent wavelengths. In some embodiments, the at least the portion ofthe 3D biological material comprises microvasculature for providing oneor more nutrients to the plurality of cells. In some embodiments, themicrovasculature is a blood microvasculature, a lymphaticmicrovasculature, or any combination thereof. In some embodiments, themicrovasculature has a cross-section from about 1 μm to about 20 μm. Insome embodiments, the 3D biological material has a thickness or diameterfrom about 100 μm to about 5 cm.

In some embodiments, the medium further comprises a plurality of beads,and wherein in (b) the at least the portion of the 3D biologicalmaterial, as formed, includes the plurality of beads. In someembodiments, the beads further comprise signaling molecules or proteins.In some embodiments, the signaling molecules or proteins promoteformation of the 3D biological material to permit organ function. Insome embodiments, the at least the portion of the 3D biological materialcomprises a cell-containing scaffold, which cell-containing scaffoldcomprises at least a subset of the plurality of cells. In someembodiments, the 3D biological material comprises cell-containingscaffolds.

In some embodiments, the cell-containing scaffolds are coupled together.In some embodiments, the cell-containing scaffolds are cohesively ormechanically coupled together. In some embodiments, the cell-containingscaffolds are mechanically coupled together through one or more membersselected from the group consisting of joints, hinges, locking joints andhinges, Velcro-like elements, springs, coils, points of stretch,interlocking loops, sockets, gears, ratchets, screw, and chain links. Insome embodiments, the cell-containing scaffolds comprise a network,wherein the network comprises a plurality of strands. In someembodiments, the plurality of strands forms a mesh structure, a gridstructure, a sheet structure, or a tube structure. In some embodiments,the individual strands of the plurality of strands have a thickness fromabout 0.1 nm to about 5 cm.

In some embodiments, subsequent to (b), the at least another portion ofthe 3D biological material is formed within the at least the portion ofthe 3D biological material. In some embodiments, the computerinstructions comprise a set of images corresponding to the 3D biologicalmaterial. In some embodiments, the computer instructions directadjustment of at least (i) one or more parameters of the at least oneenergy beam as a function of time during formation of the 3D biologicalmaterial, and/or (ii) location of a stage upon which the 3D biologicalmaterial is formed.

In some embodiments, the method further comprises subjecting at least aportion of the at least the subset of the plurality of cells todifferentiation to form the cells of the at least two different types.In some embodiments, the at least the subset of the plurality of cellscomprises cells of at least two different types. In some embodiments, in(b), the plurality of cells comprises the cells of the at least twodifferent types. In some embodiments, the at least one energy beam is amulti-photon energy beam. In some embodiments, the multi-photon energybeam is a two-photon energy beam.

In another aspect, the present disclosure provides a method of printinga three-dimensional (3D) biological material, comprising: (a) providinga media chamber comprising a first medium, wherein the first mediumcomprises a first plurality of cells and a first polymeric precursor;(b) directing at least one energy beam to the first medium in the mediachamber along at least one energy beam path in accordance with computerinstructions for printing the 3D biological material in computer memory,to subject at least a portion of the first medium in the media chamberto form a first portion of the 3D biological material; (c) providing asecond medium in the media chamber, wherein the second medium comprisesa second plurality of cells and a second polymeric precursor, whereinthe second plurality of cells is of a different type than the firstplurality of cells; and (d) directing at least one energy beam to thesecond medium in the media chamber along at least one energy beam pathin accordance with the computer instructions, to subject at least aportion of the second medium in the media chamber to form at least asecond portion of the 3D biological material.

In some embodiments, the biological material is a biologicallyfunctional tissue. In some embodiments, the method further comprises,subsequent to (d), removing a remainder of the first medium from themedia chamber to leave the first portion of the 3D biological materialin the media chamber. In some embodiments, the first portion of the 3Dbiological material left in the medium chamber is removably fixed to themedia chamber. In some embodiments, the method further comprises priorto (b), generating a point-cloud representation or lines-basedrepresentation of the 3D biological material in computer memory, andusing the point-cloud representation or lines-based representation togenerate the computer instructions.

In some embodiments, the method further comprises converting thepoint-cloud representation or lines-based representation into an imageor image set that is used to spatially modulate an incoming coherentlight source such that biological structures are projected in onedimension. In some embodiments, the method further comprises convertingthe point-cloud representation or lines-based representation into animage or image set that is used to spatially modulate an incomingcoherent light source such that biological structures are projected intwo dimensions. In some embodiments, the method further comprisesconverting the point-cloud representation or lines-based representationinto an image or image set that is used to spatially modulate anincoming coherent light source such that biological structures areprojected in three dimensions.

In some embodiments, the method further comprises converting thepoint-cloud representation or lines-based representation into an imageor image set that is used to spatially modulate at least one incomingcoherent light source such that biological structures are projected in amixture of one-dimensional, two-dimensional and/or three-dimensionalstructures. In some embodiments, the image or image set is projected ina holographic manner. In some embodiments, the image or image set isdeconstructed and reconstructed into partial elements or representativestructures prior to projection in a holographic manner.

In some embodiments, the point-cloud representation or the lines-basedrepresentation comprises multi-dimensional structural elementscorresponding to the 3D biological material. In some embodiments, thepoint-cloud representation or the lines-based representation comprisesstructural elements in two dimensions, wherein the structural elementsare associated with tissue function and/or cellular segregation. In someembodiments, the point-cloud representation or the lines-basedrepresentation comprises structural elements in three dimensions,wherein the structural elements are associated with tissue functionand/or cellular segregation.

In some embodiments, the at least one energy beam comprises coherentlight. In some embodiments, the at least one energy beam is generated bya laser. In some embodiments, the at least one energy beam is phasemodulated. In some embodiments, the at least one energy beam is phasemodulated and raster-scanned throughout the sample medium. In someembodiments, the at least a portion of the 3D biological materialcomprises microvasculature for providing one or more nutrients to theplurality of cells. In some embodiments, the microvasculature is a bloodmicrovasculature, a lymphatic microvasculature, or any combinationthereof. In some embodiments, the microvasculature has a cross-sectionfrom about 1 μm to about 20 μm. In some embodiments, the 3D biologicalmaterial has a thickness or diameter from about 100 μm to about 5 cm.

In some embodiments, the first medium and/or the second medium furthercomprise a plurality of beads, and wherein in (b) the at least theportion of the 3D biological material, as formed, includes the pluralityof beads. In some embodiments, the beads further comprise signalingmolecules or proteins. In some embodiments, the signaling molecules orthe proteins promote formation of the 3D biological material to permitorgan function. In some embodiments, the 3D biological material isprinted in a time period of at most about 350 hours. In someembodiments, the 3D biological material is printed in a time period ofat most about 72 hours. In some embodiments, the 3D biological materialis printed in a time period of at most about 12 hours.

In some embodiments, the at least the portion of the 3D biologicalmaterial comprises a cell-containing scaffold, which cell-containingscaffold comprises at least a subset of the plurality of cells. In someembodiments, the 3D biological material, as formed, includes a pluralityof cell-containing scaffolds. In some embodiments, the plurality ofcell-containing scaffolds is coupled together. In some embodiments, theplurality of cell-containing scaffolds are coupled together to form acohesive structure. In some embodiments, the plurality of thecell-containing scaffolds is mechanically coupled together. In someembodiments, the plurality of cell-containing scaffolds are mechanicallycoupled together through one or more members selected from the groupconsisting of joints, hinges, locking joints and hinges, Velcro-likeelements, springs, coils, points of stretch, interlocking loops,sockets, gears, ratchets, screw, and chain links.

In some embodiments, the cell-containing scaffolds comprise a network,wherein the network comprises a plurality of strands. In someembodiments, the plurality of strands forms a mesh structure, a gridstructure, a sheet structure, or a tube structure. In some embodiments,the plurality of strands has a thickness from about 0.1 nm to about 5cm. In some embodiments, subsequent to (d), the at least another portionof the 3D biological material is formed within the first portion of the3D biological material and/or the second portion of the 3D biologicalmaterial. In some embodiments, the first medium and/or the second mediumfurther comprise glutathione or a functional variant thereof.

In another aspect, the present disclosure provides a system for printinga three-dimensional (3D) biological material, comprising: (a) a mediachamber configured to contain a medium comprising a plurality of cellscomprising cells of at least two different types and one or more polymerprecursors; (b) at least one energy source configured to direct at leastone energy beam to the media chamber; and (c) one or more computerprocessors operatively coupled to the at least one energy source,wherein the one or more computer processors are individually orcollectively programmed to (i) receive computer instructions forprinting the 3D biological material from computer memory; and (ii)direct the at least one energy source to direct the at least one energybeam to the medium in the media chamber along at least one energy beampath in accordance with the computer instructions, to subject at least aportion of the polymer precursors to form at least a portion of the 3Dbiological material.

In some embodiments, the one or more computer processors areindividually or collectively programmed to generate a point-cloudrepresentation or lines-based representation of the 3D biologicalmaterial in computer memory, and use the point-cloud representation orlines-based representation to generate the computer instructions forprinting the 3D biological material in computer memory. In someembodiments, the one or more computer processors are individually orcollectively programmed to convert the point-cloud representation orlines-based representation into an image. In some embodiments, the oneor more computer processors are individually or collectively programmedto project the image in a holographic manner.

In some embodiments, the at least one energy source is a plurality ofenergy sources. In some embodiments, the plurality of energy sourcesdirects a plurality of the at least one energy beam. In someembodiments, the at least one energy source is a laser. In someembodiments, the at least one energy source is derived from a coherentlight source. In some embodiments, the coherent light source comprises awavelength from about 300 nm to about 5 mm. In some embodiments, the oneor more computer processors are individually or collectively programmedto direct the at least one energy source to direct the at least oneenergy beam along one or more additional energy beam paths to form atleast another portion of the 3D biological material. In someembodiments, the one or more additional energy beam paths are along an xaxis, an x and y plane, or the x, y, and z planes.

In some embodiments, the method further comprises at least one objectivelens for directing the at least one energy beam to the medium in themedia chamber. In some embodiments, the at least one objective lenscomprises a water dipping objective lens. In some embodiments, the oneor more computer processors are individually or collectively programmedto receive images of the edges of the 3D biological material. In someembodiments, the one or more computer processors are individually orcollectively programmed to direct linking of the 3D biological materialwith other tissue, which linking is in accordance with the computerinstructions. In some embodiments, the plurality of cells comprisescells of at least two different types. In some embodiments, the mediumfurther comprises glutathione or a functional variant thereof.

In another aspect, the present disclosure provides a system for printinga three-dimensional (3D) biological material, comprising: (a) a mediachamber configured to contain a medium comprising a plurality of cellsand a plurality of polymer precursors; (b) at least one energy sourceconfigured to direct at least one energy beam to the media chamber; and(c) one or more computer processors operatively coupled to the at leastone energy source, wherein the one or more computer processors areindividually or collectively programmed to (i) receive computerinstructions for printing the 3D biological material from computermemory; (ii) direct the at least one energy source to direct the atleast one energy beam to the medium in the media chamber along at leastone energy beam path in accordance with the computer instructions, tosubject at least a portion of the polymer precursors to form at least aportion of the 3D biological material; and (iii) direct the at least oneenergy source to direct the at least one energy beam to a second mediumin the media chamber along at least one energy beam path in accordancewith the computer instructions, to subject at least a portion of thesecond medium in the media chamber to form at least a second portion ofthe 3D biological material, wherein the second medium comprises a secondplurality of cells and a second polymeric precursor, wherein the secondplurality of cells is of a different type than the first plurality ofcells.

In some embodiments, the one or more computer processors areindividually or collectively programmed to generate a point-cloudrepresentation or lines-based representation of the 3D biologicalmaterial, and use the point-cloud representation or lines-basedrepresentation to generate the computer instructions for printing the 3Dbiological material in computer memory. In some embodiments, the one ormore computer processors are individually or collectively programmed toconvert the point-cloud representation or lines-based representationinto an image. In some embodiments, the one or more computer processorsare individually or collectively programmed to project the image in aholographic manner. In some embodiments, the at least one energy sourceis a plurality of energy sources. In some embodiments, the plurality ofenergy sources directs a plurality of the at least one energy beam. Insome embodiments, the at least one energy source is a laser. In someembodiments, the at least one energy source is derived from a coherentlight source.

In some embodiments, the coherent light source comprises a wavelengthfrom about 300 nm to about 5 mm. In some embodiments, the one or morecomputer processors are individually or collectively programmed todirect the least one energy source to direct the at least one energybeam along one or more additional energy beam paths to form at leastanother portion of the 3D biological material. In some embodiments, theone or more additional energy beam paths are along an x axis, an x and yplane, or the x, y, and z planes. In some embodiments, the systemfurther comprises at least one objective lens for directing the at leastone energy beam to the medium in the media chamber. In some embodiments,the at least one objective lens comprises a water dipping objectivelens. In some embodiments, the one or more computer processors areindividually or collectively programmed to receive images of the edgesof the 3D biological material. In some embodiments, the one or morecomputer processors are individually or collectively programmed todirect linking of the 3D biological material with other tissue, whichlinking is in accordance with the computer instructions. In someembodiments, the medium further comprises glutathione or a functionalvariant thereof.

In another aspect, the present disclosure provides a method for printinga three-dimensional (3D) object, comprising: directing at least oneenergy beam into a medium comprising one or more precursors, to generatethe 3D object comprising a material formed from the one or moreprecursors, wherein the at least one energy beam is directed into themedium as a 3D projection corresponding to the 3D object.

In some embodiments, the material is a polymeric material. In someembodiments, the medium comprises cells or cellular constituents. Insome embodiments, the one or more precursors are polymeric precursors.In some embodiments, the one or more precursors include one or moremetals. In some embodiments, the 3D projection is a hologram. In someembodiments, the medium further comprises glutathione or a functionalvariant thereof.

In another aspect, the present disclosure provides a method for printinga three-dimensional (3D) biological material, comprising: (a) directingat least a first energy beam into a media chamber comprising a firstmedium comprising (i) a first plurality of cells and (ii) a firstpolymeric precursor, to generate a first portion of the 3D biologicalmaterial, and; (b) directing at least a second energy beam into themedia chamber comprising a second medium comprising (i) a secondplurality of cells and (ii) a second polymeric precursor, to generate asecond portion of the 3D biological material adjacent to the firstportion of the 3D biological material.

In some embodiments, the at least first energy beam and the at leastsecond energy beam are from the same energy source. In some embodiments,the at least first energy beam and the at least second energy beam arelaser beams. In some embodiments, the cells of the first plurality ofcells and the cells of the second plurality of cells are of differenttypes. In some embodiments, the cells of the first plurality of cellsand the cells of the second plurality of cells are of the same type. Insome embodiments, the first polymeric precursor and the second polymericprecursor are different. In some embodiments, the first polymericprecursor and the second polymeric precursor are the same. In someembodiments, the first medium and/or second medium further compriseglutathione or a functional variant thereof.

The present disclosure provides methods and systems for rapid generationof multilayered vascularized tissues using spatial light modulation ofmulti-photon excitation sources. Using this approach, a method for rapidcreation of cell-containing structures is provided by layering cell-sizespecific nets with embedded mechanical and, or biological elements suchas microvasculature. The deposition of cells contained in nets ofcollagen or another biologically compatible, or inert material, is arapid, iterative, process based on a three dimensional (holographic)projection, a two-dimensional projection, and/or in any planar axis suchas x, y, x, z, or y, z, which may be combined with scanning of themulti-photon laser excitation. Three dimensional scanning,two-dimensional scanning, and raster scanning may be used simultaneouslyin various combinations to achieve rapid creation of a completestructure. The dynamic shifts between modes of laser projection allowsfor rapid generation of complex structures in a large field of view,while maintaining fine micrometer to nanometer resolution. This methodallows for rapid production of large (e.g., up to about 5 centimeters(cm)) multi-layered and small vasculature (e.g. 1-10 micrometers (μm))single-cell layered vasculature.

The present disclosure permits layering of multiple cell types in twodimensions and/or three dimensions such that tissue may be constructedin a manner that is not limited by multiple cell types, sizes, orcomplexities. In some cases, this is achieved using multiphoton (e.g.,two-photon) excitation light, as may be provided, for example, by alaser.

Another aspect of the present disclosure provides a non-transitorycomputer readable medium comprising machine executable code that, uponexecution by one or more computer processors, implements any of themethods above or elsewhere herein.

Another aspect of the present disclosure provides a system comprisingone or more computer processors and computer memory coupled thereto. Thecomputer memory comprises machine executable code that, upon executionby the one or more computer processors, implements any of the methodsabove or elsewhere herein.

Additional aspects and advantages of the present disclosure will becomereadily apparent to those skilled in this art from the followingdetailed description, wherein only illustrative embodiments of thepresent disclosure are shown and described. As will be realized, thepresent disclosure is capable of other and different embodiments, andits several details are capable of modifications in various obviousrespects, all without departing from the disclosure. Accordingly, thedrawings and description are to be regarded as illustrative in nature,and not as restrictive.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.To the extent publications and patents or patent applicationsincorporated by reference contradict the disclosure contained in thespecification, the specification is intended to supersede and/or takeprecedence over any such contradictory material.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings (also “Figure” and “FIG.” herein), of which:

FIG. 1 illustrates an embodiment of a system for rapid multi-photonprinting of a desired tissue is illustrated.

FIGS. 2A-2D illustrate example stages of the generation of a desiredtissue within the media chamber. FIG. 2A illustrates the media chambercontaining media comprising a first cell group. FIG. 2B illustrates themedia chamber containing media comprising a second cell group. FIG. 2Cillustrates delivery of pulses of the multi-photon laser beam to themedia. FIG. 2D illustrates an embodiment wherein the cell-containingscaffolding is printed along the bottom of the media chamber containingmedia.

FIGS. 3A-3C illustrate various embodiments of a laser system. FIG. 3Aillustrates an embodiment of a laser system having a single multi-photonlaser source. FIG. 3B illustrates an embodiment of a laser system havingmultiple laser lines. FIG. 3C illustrates an embodiment of a lasersystem comprising multiple laser lines, photomultipliers (PMTs), and anobjective lens.

FIGS. 4A-4C illustrate various embodiments of the printing system. FIG.4A illustrates an embodiment of the printing system comprising a beamexpander, an optical focusing lens, an additional laser focusing lens,and no axicon or TAG lens. FIG. 4B illustrates an embodiment of theprinting system comprising a beam expander, an optical focusing lens, anadditional laser focusing lens, and an axicon or TAG lens. FIG. 4Cillustrates a Z-step projection printing setup comprising a single SLMor DMD for 2D, x, y sheet or hologram projection for printing aroundcells and resultant structures printed with given Z-steps.

FIGS. 5A-5B illustrate various embodiments of the multi-photon tissueprint head. FIG. 5A illustrates an embodiment of the multi-photon tissueprint head comprising a single, upright objective lens. FIG. 5Billustrates an embodiment of the multi-photon tissue print head havinginverted optics for imaging structures.

FIGS. 6A-6B illustrate embodiments of a removable and attachable fiberoptic cable accessory. FIG. 6A illustrates the fiber optic cableaccessory and fiber optic cable. FIG. 6B illustrates the fiber opticcable accessory being used to print the desired complex tissuestructure.

FIG. 7 illustrates an embodiment wherein the print-head optics includesat least three objectives, wherein each objective includes a fiber opticcable accessory directed into a single media chamber.

FIG. 8 illustrates an embodiment wherein the print-head optics includesat least six objectives, wherein each objective includes a fiber opticcable accessory directed into a separate media chamber such as aseparate well of a multi-well plate.

FIG. 9 illustrates embodiments of print-head optics having an array ofobjectives acting as print heads.

FIG. 10 illustrates objectives programmed to move over the multi-wellplate in X and Y directions to deliver the laser beam projections intoeach well.

FIG. 11 illustrates an example net structure formed from polymerizablematerial.

FIGS. 12A-12B illustrate various embodiments of net structures. FIG. 12Aillustrates a net comprised of strands having a thickness of about 0.1micrometers. FIG. 12B illustrates a net comprised of strands having athickness of approximately 5 micrometers.

FIG. 13 illustrates rounded cells temporarily trapped within a net.

FIG. 14 illustrates a first net and a second net disposed near eachother so that cells are able to move through the apertures and engageunder physiological conditions.

FIGS. 15A-15C illustrate a method of creating areas of such structuralfeatures within a net structure 500. FIG. 15A illustrates the generationof a net structure. FIG. 15B illustrates a second projection of amulti-photon laser beam from the laser beam targeting specificcoordinates within the net structure. FIG. 15C illustrates the final netstructure having the various points of reinforcement at thepre-determined intersections of the net strands.

FIGS. 16A-16D illustrate another method of creating areas of suchstructural features within a net. FIG. 16A shows the generation of a netstructure by projecting the multi-photon laser beam from the optics ofthe multi-photon tissue printing print-head into the media. FIG. 16Billustrates a second projection of a multi-photon laser beam targetingspecific coordinates within the net structure. FIG. 16C illustrates thefinal net structure having the reinforced zig-zag shaped structuralfeature. FIG. 16D illustrates thicker net regions directing structuraldeformation.

FIGS. 17A-17B illustrate the use of structural features within a net tocause the tissue structure to fold or wrinkle. FIG. 17A illustrates anet structure formed within the media wherein the net structure includesstructural reinforcements. FIG. 17B illustrates first net strand and thesecond net strand drawn towards each other.

FIGS. 18A-18B illustrate the use of structural features within a net tocause the tissue structure to fold or wrinkle. FIG. 18A illustrates thedownward motion of the cells (indicated by arrows) as the cells move andcommunicate, to form the folds. FIG. 18B illustrates the cells havingformed the folds between the first net strand, the second net strand,and the third net strand drawing the first net strand and the second netstrand toward each other.

FIGS. 19A-19C illustrate another embodiment of a net having increasedareas of thickness. FIG. 19A illustrates a net structure formed withinmedia wherein the net structure includes a first structuralreinforcement, a second structural reinforcement, and a third structuralreinforcement. FIG. 19B illustrates the first net strand and the secondnet strand drawn toward each other. FIG. 19C provides a side view of thetissue showing the first unreinforced portion, the second unreinforcedportion, and the third unreinforced portion drawn together, formingfolds or wrinkles.

FIG. 20 illustrates a net structure having a high density net regionsurrounded by a low density net region.

FIG. 21 illustrates another embodiment wherein variations in density ofthe net structure guide movement and interactions of cells.

FIG. 22 illustrates another embodiment wherein variations in density ofthe net structure guide movement and interactions of cells to make athree-dimensional tissue structure.

FIGS. 23A-23E illustrate textured elements along net strands which maypromote cell adhesion or attraction. FIG. 23A illustrates a firstexample of a textured element. FIG. 23B illustrates a second example ofa textured element. FIG. 23C illustrates a third example of a texturedelement. FIG. 23D illustrates a fourth example of a textured element.FIG. 23E illustrates a fifth example of a textured element.

FIGS. 24A-24B illustrate textured elements along net strands which maypromote cell adhesion or attraction. FIG. 24A illustrates a sixthexample of a textured element. FIG. 24B illustrates a seventh example ofa textured element.

FIG. 25 illustrates yet another example of textured elements along netstrands which may promote cell adhesion or attraction.

FIG. 26 illustrates an embodiment of a net having a cleavage site.

FIGS. 27A-27B illustrate an embodiment of a mechanical elementcomprising a pivot joint. FIG. 27A illustrates a pivot joint comprisinga first protrusion and a second protrusion. FIG. 27B illustrates thefirst protrusion being attached to a first net structure and the secondprotrusion being attached to a second net structure.

FIGS. 28A-28B illustrate an embodiment of a mechanical elementcomprising a ball-and-socket joint. FIG. 28A illustrates aball-and-socket joint comprising a first protrusion having a roundedball head and a second protrusion having a concave socket head. FIG. 28Billustrates a ball-and-socket joint that is printed so that the firstprotrusion is attached to a first net structure and the secondprotrusion is attached to a second net structure.

FIGS. 29A-29B illustrate an embodiment of a mechanical elementcomprising a saddle joint. FIG. 29A illustrates a saddle jointcomprising a first protrusion and a second protrusion having a having asaddle-shaped indentation. FIG. 28B illustrates a saddle joint that isprinted so that the first protrusion and second protrusions are attachedto the net structures.

FIG. 30 illustrates an embodiment of a socket joint comprising a firstprotrusion and a second protrusion having socket-shaped cavities.

FIG. 31 illustrates an embodiment of a socket joint that is printed sothat the first protrusion and the second protrusion are attached to netstructures.

FIG. 32 illustrates an embodiment of threaded joint comprising a firstprotrusion having a first head and a second head with socket-shapedcavities having grooves and threads.

FIG. 33 illustrates an embodiment of threaded joint that is printed sothat the first protrusion and second protrusion are attached to netstructures.

FIGS. 34A-34B illustrate an embodiment of a mechanical elementcomprising a coil or spring. FIG. 34A illustrates an embodiment of aspring. FIG. 34B illustrates an embodiment of spring that is printed sothat its ends are attached to the net structures.

FIGS. 35A-35B illustrate a mechanical element comprising a chain. FIG.35A illustrates an embodiment of a chain comprising two ends and fourlinks. FIG. 35B illustrates an embodiment of a chain that is printed sothat its ends are attached to net structures.

FIGS. 36A-36B illustrate an embodiment of a mechanical elementcomprising a hooking joint. FIG. 36A illustrates an embodiment of ahooking joint having a curved shape. FIG. 36B illustrates an embodimentof hooking joint that is printed so that the hooks are attached to thenet structures.

FIGS. 37A-37C illustrate an embodiment of a mechanical elementcomprising a hook-and-loop joint which functions in a manner similar toVelcro®. FIG. 37A illustrates an embodiment of a hook-and-loop jointcomprising a hook surface that is mateable with a loop surface. FIG. 37Billustrates an embodiment of the hook-and-loop joint being disengaged.FIG. 37C illustrates an embodiment of a hook-and-loop joint that isprinted so that the hook surface is attached to the net structures.

FIGS. 38A-38C illustrate an embodiment of a mechanical elementcomprising a hinge. FIG. 38A illustrates an embodiment of a hingecomprising brackets. FIG. 38B illustrates a rod extending through andjoining two brackets. FIG. 38C illustrates an embodiment of a hinge thatis printed so that the brackets are attached to net structures.

FIG. 39 illustrates an embodiment of a tissue structure comprised ofcells captured in a net wherein the net is looping due to the presenceof mechanical elements.

FIG. 40 illustrates an embodiment of a tissue structure comprised ofcells captured in a net wherein the net is twisting due to the presenceof mechanical elements.

FIGS. 41A-41B illustrate an embodiment designed to induce cell-cellinteractions between two separate cell groups located in two separatenet structures. FIG. 41A illustrates two nets having an edge. FIG. 41Billustrates the cells being held along the edges, favoring theoccurrence of cell-cell interactions with each other.

FIGS. 42A-42B illustrate embodiments of variable density nets can beused to generate cell strands. FIG. 42A illustrates net structurecomprising a longitudinal region wherein the first apertures are sizedto trap particular cells and the surrounding second apertures are sizedto exclude cells. FIG. 42B illustrates the creation of a cell strandusing variable density nets.

FIG. 43 shows a computer control system that is programmed or otherwiseconfigured to implement methods provided herein.

FIG. 44 illustrates the optical components and optical path of anembodiment of the printing system without temporal focusing.

FIG. 45 illustrates the optical components and optical path of anadditional embodiment of the printing system with temporal focusing.

FIG. 46 illustrates the optical components and optical path of yetanother embodiment of the printing system without temporal focusing.

FIG. 47 illustrates a light detection system.

FIGS. 48A-48E show examples of a cellularized, three-dimensional (3D),impermeable microvasculature structure generated by holographicprinting. FIG. 48A shows a top-down view of the 3D microvasculature.FIG. 48B shows a top-down view of the outer tube of the 3Dmicrovasculature. FIG. 48C shows a completed 3D microvasculaturestructure. FIG. 48D shows a fluorescent image of three microvasculaturestructures encapsulating cells. FIG. 48E shows a bright field image ofthree microvasculature structures encapsulating cells after five days ofholographic printing.

FIGS. 49A-49H show an exemplary process of the generation of acell-containing structure using holographic printing. FIG. 49A shows acomputer generated three-dimensional (3D) image of a cell-containingstructure. FIG. 49B shows a point-cloud representation of the 3D imageof the cell-containing structure. FIG. 49C shows a hologramcorresponding to the point-cloud representation of the 3D image of thecell-containing structure. FIG. 49D illustrates the computer printingsystem. FIG. 49E shows an image of a cluster of cells suspended inliquid print media. FIG. 49F shows an image of the same cluster ofliving cells after three dimensional printing of the point-cloudrepresentation. FIG. 49G shows a cut-away image showing cells within theprinted, 3D cell-containing structure. FIG. 49H shows a representativeimage of the completed 3D cell-containing structure after printing.

FIGS. 50A-50C show images of the holographic printing of the “StanfordBunny.” FIG. 50A shows a computer generated three-dimensional (3D) imageof the “Stanford Bunny.” FIG. 50B shows a top-down view of the computergenerated 3D image of the “Stanford Bunny.” FIG. 50C shows arepresentative 3D print of the “Stanford Bunny” as imaged using inbright-field microscopy.

FIGS. 51A-51B show graphs of a two-photon laser beam exposure time (inmilliseconds) vs. laser power (Watts) corresponding to holographicprinting of two different formulations. FIG. 51A shows the threshold forprinting in Formulation A. FIG. 51B shows the threshold for printing inFormulation B.

FIGS. 52A-52C show targeted single cell encapsulation using holographicprinting. FIG. 52A shows a plurality of encapsulated cells andnon-encapsulated cells suspended in print media. FIG. 52B showszoomed-in images of a plurality of encapsulated cells. FIG. 52C showszoomed-in images of a plurality of non-encapsulated cells.

FIG. 53 shows an expanded laser beam projecting a hologram.

FIGS. 54A-54D illustrate different laser printing modes. FIG. 54Aillustrates a single photon laser beam projection into a media chambercontaining a photosensitive print medium. FIG. 54B illustrates amulti-photon absorption process. FIG. 54C illustrates a representativegraphic of wavefront shaping to produce a hologram. FIG. 54D illustratesa complete image projection (i.e., a 3D hologram) in multiple planesallowing for the holographic printing of a complex structure.

FIGS. 55A-55F show the holographic printing of a sphere within apreviously printed 3D microvascular structure. FIG. 55A illustrates aprinted microvasculature structure. FIG. 55B shows an image of a printedmicrovasculature structure. FIG. 55C illustrates the use of amulti-photon laser beam to project a hologram of a sphere into the lumenof the 3D microvasculature structure. FIG. 55D shows an image of the 3Dmicrovasculature structure exactly when a multi-photon laser beam wasused to project a hologram of a sphere into the lumen of the 3Dmicrovasculature structure. FIG. 55E illustrates a sphere inside thelumen of the microvasculature structure. FIG. 55F shows the sphere(outlined by the dashed circle) was deposited within the lumen of themicrovasculature structure without disrupting it.

FIGS. 56A-56B show images of a polymeric vasculature bed printed usingthe methods and systems provided herein. FIG. 56A shows an image of thevasculature bed during the holographic printing process. FIG. 56B showsan image of the vasculature bed after the holographic printing processis completed.

DETAILED DESCRIPTION

While various embodiments of the invention have been shown and describedherein, it will be obvious to those skilled in the art that suchembodiments are provided by way of example only. Numerous variations,changes, and substitutions may occur to those skilled in the art withoutdeparting from the invention. It should be understood that variousalternatives to the embodiments of the invention described herein may beemployed.

The terminology used herein is for the purpose of describing particularcases only and is not intended to be limiting. As used herein, thesingular forms “a”, “an” and “the” are intended to include the pluralforms as well, unless the context clearly indicates otherwise.Furthermore, to the extent that the terms “including”, “includes”,“having”, “has”, “with”, or variants thereof are used in either thedetailed description and/or the claims, such terms are intended to beinclusive in a manner similar to the term “comprising.”

The term “about” or “approximately” refers to an amount that is near thestated amount by about 10%, 5%, or 1%, including increments therein. Forexample, “about” or “approximately” can mean a range including theparticular value and ranging from 10% below that particular value andspanning to 10% above that particular value.

The term “biological material,” as used herein, generally refers to anymaterial that may serve a chemical or biological function. Biologicalmaterial may be biologically functional tissue or functional tissue,which may be a biological structure that is capable of serving, orserving, a biomechanical or biological function. Biologically functionaltissue may comprise cells that are within diffusion distance from eachother, comprises at least one cell type wherein each cell is withindiffusion distance of a capillary or vascular network component,facilitates and/or inhibits the fulfillment of protein function, or anycombination thereof. Biologically functional tissue may be at least aportion of tissue or an organ, such as a vital organ. In some examples,the biological material may be used for drug development, such as, forexample, screening multiple cells or tissue with different therapeuticagents.

Biological material may include a matrix, such as a polymeric matrix,including one or more other types of material, such as cells. Biologicalmaterial may be in various shapes, sizes or configurations. In someinstances, biological material may be consumable by a subject (e.g., ananimal), such as meat or meat-like material.

The term “three-dimensional printing” (also “3D printing”), as usedherein, generally refers to a process or method for generating a 3D part(or object). Such process may be used to form a 3D part (or object),such as a 3D biological material.

The term “energy beam,” as used herein, generally refers to a beam ofenergy. The energy beam may be a beam of electromagnetic energy orelectromagnetic radiation. The energy beam may be a particle beam. Anenergy beam may be a light beam (e.g., gamma waves, x-ray, ultraviolet,visible light, infrared light, microwaves, or radio waves). The lightbeam may be a coherent light beam, as may be provided by lightamplification by stimulated emission of radiation (“laser”). In someexamples, the light beam is generated by a laser diode or a multiplediode laser.

The present disclosure provides methods and systems for printing athree-dimensional (3D) biological material. In an aspect, a method forprinting the 3D biological material comprises providing a media chambercomprising a medium comprising (i) a plurality of cells and (ii) one ormore polymer precursors. Next, at least one energy beam may be directedto the medium in the media chamber along at least one energy beam paththat is patterned into a 3D projection in accordance with computerinstructions for printing the 3D biological material in computer memory.This may form at least a portion of the 3D biological materialcomprising (i) at least a subset of the plurality of cells, which atleast the subset of the plurality of cells comprises cells of at leasttwo different types, and (ii) a polymer formed from the one or morepolymer precursors.

Methods and systems of the present disclosure may be used to printmultiple layers of a 3D object, such as a 3D biological material, at thesame time. Such 3D object may be formed of a polymeric material, ametal, metal alloy, composite material, or any combination thereof. Insome examples, the 3D object is formed of a polymeric material, in somecases including biological material (e.g., one or more cells or cellularcomponents). In some cases, the 3D object may be formed by directing anenergy beam (e.g., a laser) as a 3D projection (e.g., hologram) to oneor more precursors of the polymeric material, to induce polymerizationand/or cross-linking to form at least a portion of the 3D object. Thismay be used to form multiple layers of the 3D object at the same time.

As an alternative, the 3D object may be formed of a metal or metalalloy, such as, e.g., gold, silver, platinum, tungsten, titanium, or anycombination thereof. In such a case, the 3D object may be formed bysintering or melting metal particles, as may be achieved, for example,by directing an energy beam (e.g., a laser beam) at a powder bedcomprising particles of a metal or metal alloy. In some cases, the 3Dobject may be formed by directing such energy beam as a 3D projection(e.g., hologram) into the powder bed to facilitate sintering or meltingof particles. This may be used to form multiple layers of the 3D objectat the same time. The 3D object may be formed of an organic materialsuch as graphene. The 3D object may be formed of an inorganic materialsuch as silicone. In such cases, the 3D object may be formed bysintering or melting organic and/or inorganic particles, as may beachieved, for example, by directing an energy beam (e.g., a laser beam)at a powder bed comprising particles of an organic and/or inorganicmaterial. In some cases, the 3D object may be formed by directing suchenergy beam as a 3D projection (e.g., hologram) into the powder bed tofacilitate sintering or melting of organic and/or inorganic particles.

The depth of the energy beam penetration may be dictated by theinteraction of the beam wavelength and the electron field of a givenmetal, metal alloy, inorganic material, and/or organic material. Theorganic material may be graphene. The inorganic material may besilicone. These particles may be functionalized or combined in to allowfor greater interaction or less interaction with a given energy beam.

In some examples, the at least one energy beam is at least 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, or more energy beams. The atleast one energy beam may be or include coherent light. In some cases,the at least one energy beam is a laser beam.

The at least one energy beam may be directed as an image or image set.The image may be fixed with time or changed with time. The at least oneenergy beam may be directed as a video.

The computer instructions may correspond to a computer model orrepresentation of the 3D biological material. The computer instructionsmay be part of the computer model. The computer instructions maycomprise a set of images corresponding to the 3D biological material.

The at least one energy beam may be directed as a holographic image orvideo. This may enable different points in the medium to be exposed tothe at least one energy beam at the same time, to, for example, induceformation of a polymer matrix (e.g., by polymerization) at multiplelayers at the same time. In some cases, a 3D image or video may beprojected into the medium at different focal points using, e.g., aspatial light modulator (SLM).

The computer instructions may include and/or direct adjustment of one ormore parameters of the at least one energy beam as a function of timeduring formation of the 3D biological material, such as, for example,application of power to a source of the at least one energy beam (e.g.,laser on/off). Such adjustment may be made in accordance with an imageor video (e.g., holographic image or video) corresponding to the 3Dbiological material. Alternatively or in addition to, the computerinstructions may include and/or direct adjustment of a location of astage upon which the 3D biological material is formed.

In some cases, during or subsequent to formation of the 3D biologicalmaterial, at least a portion of the at least the subset of the pluralityof cells may be subjected to differentiation to form the cells of the atleast two different types. This may be employed, for example, byexposing the cells to an agent or subjecting the cells to a conditionthat induces differentiation. Alternatively or in addition to, the cellsmay be subjected to de-differentiation.

Another aspect of the present disclosure provides a method for printinga 3D biological material, providing a media chamber comprising a firstmedium. The first medium may comprise a first plurality of cells and afirst polymeric precursor. At least one energy beam may be directed tothe first medium in the media chamber along at least one energy beampath in accordance with computer instructions for printing the 3Dbiological material, to subject at least a portion of the first mediumin the media chamber to form a first portion of the 3D biologicalmaterial. Next, a second medium may be provided in the media chamber.The second medium may comprise a second plurality of cells and a secondpolymeric precursor. The second plurality of cells may be of a differenttype than the first plurality of cells. Next, at least one energy beammay be directed to the second medium in the media chamber along at leastone energy beam path in accordance with the computer instructions, tosubject at least a portion of the second medium in the media chamber toform at least a second portion of the 3D biological material.

In another aspect of the present disclosure, a system for printing a 3Dbiological material comprises a media chamber configured to contain amedium comprising a plurality of cells comprising cells of at least twodifferent types and one or more polymer precursors; at least one energysource configured to direct at least one energy beam to the mediachamber; and one or more computer processors operatively coupled to theat least one energy source, wherein the one or more computer processorsare individually or collectively programmed to (i) receive computerinstructions for printing the 3D biological material from computermemory; and (ii) direct the at least one energy source to direct the atleast one energy beam to the medium in the media chamber along at leastone energy beam path in accordance with the computer instructions, tosubject at least a portion of the polymer precursors to form at least aportion of the 3D biological material.

In another aspect, a system for printing a 3D biological material,comprising: a media chamber configured to contain a medium comprising aplurality of cells and a plurality of polymer precursors; at least oneenergy source configured to direct at least one energy beam to the mediachamber; and one or more computer processors operatively coupled to theat least one energy source, wherein the one or more computer processorsare individually or collectively programmed to (i) receive computerinstructions for printing the 3D biological material from computermemory; (ii) direct the at least one energy source to direct the atleast one energy beam to the medium in the media chamber along at leastone energy beam path in accordance with the computer instructions, tosubject at least a portion of the polymer precursors to form at least aportion of the 3D biological material; and (iii) direct the at least oneenergy source to direct the at least one energy beam to a second mediumin the media chamber along at least one energy beam path in accordancewith the computer instructions, to subject at least a portion of thesecond medium in the media chamber to form at least a second portion ofthe 3D biological material, wherein the second medium comprises a secondplurality of cells and a second polymeric precursor, wherein the secondplurality of cells is of a different type than the first plurality ofcells.

In another aspect of the present disclosure, methods for printing athree-dimensional (3D) object, may comprise directing at least oneenergy beam into a medium comprising one or more precursors, to generatethe 3D object comprising a material formed from the one or moreprecursors, wherein the at least one energy beam is directed into themedium as a 3D projection corresponding to the 3D object.

In another aspect, methods for printing a three-dimensional (3D)biological material, may comprise directing at least one energy beamto: 1) a first medium comprising a first plurality of cells and a firstpolymeric precursor, and 2) a second medium comprising a secondplurality of cells and a second polymeric precursor, to generate a firstportion of the 3D biological material and a second portion of the 3Dbiological material.

Referring to FIG. 1, an embodiment of a system 100 for rapidmulti-photon printing of a desired tissue is illustrated. Here, thesystem 100 comprises a laser printing system 110 driven by a solid-modelcomputer-aided design (CAD) modeling system 112. In this embodiment, theCAD modeling system 112 comprises a computer 114 which controls thelaser printing system 110 based on a CAD model of the desired tissue andadditional parameters. The laser printing system 110 comprises a lasersystem 116 in communication with a multi-photon tissue printingprint-head 118 which projects waveforms of a multi-photon laser beam 120into a media chamber 122 to match the desired structure in complete orin specific parts. The multi-photon tissue print-head 118 includes atleast one objective lens 124 that delivers the multi-photon laser beam120 in the lateral and axial planes of the media chamber 122 to providea two-dimensional and/or three dimensional and thus holographicprojection of the CAD modeled tissue within the media chamber 122. Theobjective lens 124 may be a water-immersion objective lens, an airobjective lens, or an oil-immersion objective lens. Two dimensional andthree dimensional holographic projections may be generatedsimultaneously and projected into different regions by lens control. Themedia chamber 122 contains media comprised of cells, polymerizablematerial, and culture medium. The polymerizable material may comprisepolymerizable monomeric units that are biologically compatible,dissolvable, and, in some cases, biologically inert. The monomeric units(or subunits) may polymerize, cross-link, or react in response to themulti-photon laser beam 120 to create cell containing structures, suchas cell matrices and basement membrane structures, specific to thetissue to be generated. The monomeric units may polymerize and/orcross-link to form a matrix. In some cases, the polymerizable monomericunits may comprise mixtures of collagen with other extracellular matrixcomponents including but not limited to elastin and hyaluronic acid tovarying percentages depending on the desired tissue matrix.

Non-limiting examples of extracellular matrix components used to createcell containing structures may include proteoglycans such as heparansulfate, chondroitin sulfate, and keratan sulfate, non-proteoglycanpolysaccharide such as hyaluronic acid, collagen, and elastin,fibronectin, laminin, nidogen, or any combination thereof. Theseextracellular matrix components may be functionalized with acrylate,diacrylate, methacrylate, cinnamoyl, coumarin, thymine, or otherside-group or chemically reactive moiety to facilitate cross-linkinginduced directly by multi-photon excitation or by multi-photonexcitation of one or more chemical doping agents. In some cases,photopolymerizable macromers and/or photopolymerizable monomers may beused in conjunction with the extracellular matrix components to createcell-containing structures. Non-limiting examples of photopolymerizablemacromers may include polyethylene glycol (PEG) acrylate derivatives,PEG methacrylate derivatives, and polyvinyl alcohol (PVA) derivatives.In some instances, collagen used to create cell containing structure maybe fibrillar collagen such as type I, II, III, V, and XI collagen, facitcollagen such as type IX, XII, and XIV collagen, short chain collagensuch as type VIII and X collagen, basement membrane collagen such astype IV collagen, type VI collagen, type VII collagen, type XIIIcollagen, or any combination thereof.

Specific mixtures of monomeric units can be created to alter the finalproperties of the polymerized biogel. This base print mixture maycontain other polymerizable monomers that are synthesized and not nativeto mammalian tissues, comprising a hybrid of biologic and syntheticmaterials. An example mixture may comprise about 0.4% w/v collagenmethacrylate plus the addition of about 50% w/v polyethylene glycoldiacrylate (PEGDA). Photoinitiators to induce polymerization may bereactive in the ultraviolet (UV), infrared (IR), or visible light range.Examples of two such photo initiators are Eosin Y (EY) andtriethanolamine (TEA), that when combined may polymerize in response toexposure to visible light (e.g., wavelengths of about 390 to 700nanometers). Non-limiting examples of photoinitiators may includeazobisisobutyronitrile (AIBN), benzoin derivatives, benziketals,hydroxyalkylphenones, acetophenone derivatives, trimethylolpropanetriacrylate (TPT), acryloyl chloride, benzoyl peroxide, camphorquinone,benzophenone, thioxanthones, and2-hydroxy-1-[4-(hydroxyethoxy)phenyl]-2-methyl-1-propanone.Hydroxyalkylphenones may include4-(2-hydroxyethylethoxy)-phenyl-(2-hydroxy-2-methyl propyl) ketone(Irgacure® 295), 1-hidroxycyclohexyl-1-phenyl ketone (Irgacure® 184) and2,2-dimethoxy-2-phenylacetophenone (Irgacure® 651). Acetophenonederivatives may include 2,2-dimethoxy-2-phenylacetophenone (DMPA).Thioxanthones may include isopropyl thioxanthone.

Specific mixtures of monomeric units of biological materials can becreated to alter the final properties of the polymerized biogel, anexample mixture may include about 1 mg/mL type I collagen-methacrylate,about 0.5 mg/mL type III collagen, about 0.2 mg/mL methacrylatedhyaluronic acid, about 0.1% Eosin Y, and about 0.1% triethanolamine.

In some cases, the polymerized biogel may comprise at least about 0.01%of a photoinitiator. In some cases, the polymerized biogel may compriseabout 10% of a photoinitiator or more. In some cases, the polymerizedbiogel comprises about 0.1% of a photoinitiator. In some cases, thepolymerized biogel may comprise about 0.01% to about 0.05%, about 0.01%to about 0.1%, about 0.01% to about 0.2%, about 0.01% to about 0.3%,about 0.01% to about 0.4%, about 0.01% to about 0.5%, about 0.01% toabout 0.6%, about 0.7% to about 0.8%, about 0.9% to about 1%, about0.01% to about 2%, about 0.01% to about 3%, about 0.01%% to about 4%,about 0.01% to about 5%, about 0.01% to about 6%, about 0.01% to about7%, about 0.01% to about 8%, about 0.01% to about 9%, or about 0.01% toabout 10% of a photoinitiator.

The polymerized biogel may comprise about 0.05% of a photoinitiator. Thepolymerized biogel may comprise 0.1% of a photoinitiator. Thepolymerized biogel may comprise about 0.2% of a photoinitiator. Thepolymerized biogel may comprise about 0.3% of a photoinitiator. Thepolymerized biogel may comprise about 0.4% of a photoinitiator. Thepolymerized biogel may comprise about 0.5% of a photoinitiator. Thepolymerized biogel may comprise about 0.6% of a photoinitiator. Thepolymerized biogel may comprise about 0.7% of a photoinitiator. Thepolymerized biogel may comprise about 0.8% of a photoinitiator. Thepolymerized biogel may comprise about 0.9% of a photoinitiator. Thepolymerized biogel may comprise about 1% of a photoinitiator. Thepolymerized biogel may comprise about 1.1% of a photoinitiator. Thepolymerized biogel may comprise about 1.2% of a photoinitiator. Thepolymerized biogel may comprise about 1.3% of a photoinitiator. Thepolymerized biogel may comprise about 1.4% of a photoinitiator. Thepolymerized biogel may comprise about 1.5% of a photoinitiator. Thepolymerized biogel may comprise about 1.6% of a photoinitiator. Thepolymerized biogel may comprise about 1.7% of a photoinitiator. Thepolymerized biogel may comprise about 1.8% of a photoinitiator. Thepolymerized biogel may comprise about 1.9% of a photoinitiator. Thepolymerized biogel may comprise about 2% of a photoinitiator. Thepolymerized biogel may comprise about 2.5% of a photoinitiator. Thepolymerized biogel may comprise about 3% of a photoinitiator. Thepolymerized biogel may comprise about 3.5% of a photoinitiator. Thepolymerized biogel may comprise about 4% of a photoinitiator. Thepolymerized biogel may comprise about 4.5% of a photoinitiator. Thepolymerized biogel may comprise about 5% of a photoinitiator. Thepolymerized biogel may comprise about 5.5% of a photoinitiator. Thepolymerized biogel may comprise about 6% of a photoinitiator. Thepolymerized biogel may comprise about 6.5% of a photoinitiator. Thepolymerized biogel may comprise about 7% of a photoinitiator. Thepolymerized biogel may comprise about 7.5% of a photoinitiator. Thepolymerized biogel may comprise about 8% of a photoinitiator. Thepolymerized biogel may comprise about 8.5% of a photoinitiator. Thepolymerized biogel may comprise about 9% of a photoinitiator. Thepolymerized biogel may comprise about 9.5% of a photoinitiator. Thepolymerized biogel may comprise about 10% of a photoinitiator.

In some cases, the polymerized biogel may comprise at least about 10% ofa photopolymerizable macromer and/or photopolymerizable monomer. In somecases, the polymerized biogel may comprise about 99% or more of aphotopolymerizable macromer and/or photopolymerizable monomer. In somecases, the polymerized biogel may comprise about 50% of aphotopolymerizable macromer and/or photopolymerizable monomer. In somecases, the polymerized biogel may comprise about 10% to about 15%, about10% to about 20%, about 10% to about 25%, about 10% to about 30%, about10% to about 35%, about 10% to about 40%, about 10% to about 45%, about10% to about 50%, about 10% to about 55%, about 10% to about 60%, about10% to about 65%, about 10% to about 70%, about 10% to about 75%, about10% to about 80%, about 10% to about 85%, about 10% to about 90%, about10% to about 95%, or about 10% to about 99% of a photopolymerizablemacromer and/or photopolymerizable monomer.

The polymerized biogel may comprise about 10% of a photopolymerizablemacromer and/or photopolymerizable monomer. The polymerized biogel maycomprise about 15% of a photopolymerizable macromer and/orphotopolymerizable monomer. The polymerized biogel may comprise about20% of a photopolymerizable macromer and/or photopolymerizable monomer.The polymerized biogel may comprise about 25% of a photopolymerizablemacromer and/or photopolymerizable monomer. The polymerized biogel maycomprise about 30% of a photopolymerizable macromer and/orphotopolymerizable monomer. The polymerized biogel may comprise about35% of a photopolymerizable macromer and/or photopolymerizable monomer.The polymerized biogel may comprise about 40% photopolymerizablemacromer and/or photopolymerizable monomer. The polymerized biogel maycomprise about 45% of a photopolymerizable macromer and/orphotopolymerizable monomer. The polymerized biogel may comprise about50% of a photopolymerizable macromer and/or photopolymerizable monomer.The polymerized biogel may comprise about 55% of a photopolymerizablemacromer and/or photopolymerizable monomer. The polymerized biogel maycomprise about 60% of a photopolymerizable macromer and/orphotopolymerizable monomer. The polymerized biogel may comprise about65% of a photopolymerizable macromer and/or photopolymerizable monomer.The polymerized biogel may comprise about 70% of a photopolymerizablemacromer and/or photopolymerizable monomer. The polymerized biogel maycomprise about 75% of a photopolymerizable macromer and/orphotopolymerizable monomer. The polymerized biogel may comprise about80% of a photopolymerizable macromer and/or photopolymerizable monomer.The polymerized biogel may comprise about 85% of a photopolymerizablemacromer and/or photopolymerizable monomer. The polymerized biogel maycomprise about 90% of a photopolymerizable macromer and/orphotopolymerizable monomer. The polymerized biogel may comprise about95% of a photopolymerizable macromer and/or photopolymerizable monomer.The polymerized biogel may comprise about 96% of a photopolymerizablemacromer and/or photopolymerizable monomer. The polymerized biogel maycomprise about 97% of a photopolymerizable macromer and/orphotopolymerizable monomer. The polymerized biogel may comprise about98% of a photopolymerizable macromer and/or photopolymerizable monomer.The polymerized biogel may comprise about 99% of a photopolymerizablemacromer and/or photopolymerizable monomer.

Two-photon absorption is non-linear and cannot be accurately predictedor calculated based on single photon absorption properties of achemical. A photo-reactive chemical may have a peak, two-photonabsorption at or around double the single photon absorption or beslightly-redshifted in absorption spectra. Therefore, wavelengths at orabout 900 nanometers through about 1400 nanometers may be used forpolymerization of monomeric materials by exciting mixtures of catalystsof the polymerization reaction, for example EY or TEA. Single wavelengthpolymerization may be sufficient for creating all structural elements,however to further speed up the printing process, multiple wavelengthsmay be employed simultaneously through the same printing apparatus andinto the same printing chamber.

Premixing or pre-reacting of polymerizable monomeric units withcatalysts comprising differing absorption bands may allow for printingat different wavelengths to form different substrate-based structuralelements simultaneously within the media chamber 122. Thus, certainstructural elements may be generated by tuning the excitation wavelengthof the laser to a particular wavelength, and then other structuralelements may be generated around the existing elements by tuning anotheror the same laser to a different excitation wavelength that may interactwith a distinct photoinitiator that initiates polymerization of onematerial base with greater efficiency. Likewise, different wavelengthsmay be used for different structural elements, wherein increasedrigidity is desired in some locations and soft or elastic structures aredesired in other locations. Because of the different physical propertiesof polymerizable materials this may allow for potentially more rigid,soft, or elastic structures to be created in the same print step withthe same cells by simply tuning the excitation wavelength of the laserelectronically, by switching between different lasers, or bysimultaneously projecting two different wavelengths.

FIGS. 2A-2C illustrate example stages of the generation of a desiredtissue within the media chamber 122. FIG. 2A illustrates the mediachamber 122 containing media 126 comprised of a first cell group,polymerizable material and culture medium. In this embodiment, pulses ofthe multi-photon laser beam 120 may be delivered to the media 126according to the CAD model corresponding to the vascular structure andmicrovasculature of the desired tissue. In some instances, the firstcell group may comprise vascular and/or microvascular cells includingbut not limited to endothelial cells, microvascular endothelial cells,pericytes, smooth muscle cells, fibroblasts, endothelial progenitorcells, stem cells, or any combination thereof. Thus, portions of themedia 126 may polymerize, cross-link or react to form cell-containingscaffolding 128 representing the vasculature and microvasculature of thedesired tissue. In this embodiment, the media 126 may then be drainedthrough a first port 130 a, a second port 130 b, a third port 130 c, afourth port 130 d, and a fifth port 130 e to remove the first cell groupand associated media. In some instances, the media chamber 122 maycomprise at least one port. In some instances, the media chamber 122 maycomprise a plurality of ports ranging from at least one port to 100ports at most. The media chamber 122 may comprise at least two ports.The media chamber 122 may comprise at least three ports. The mediachamber 122 may comprise at least four ports. The media chamber 122 maycomprise at least five ports.

Referring to FIG. 2B, the media chamber 122 may be filled with media 126containing a second cell group, polymerizable material and culturemedium through ports 130. This second cell group may be used to generatetissue structures around the existing cell-containing scaffolding 128.In some instances, the cell-containing scaffolding 128 may be a vascularscaffold. The printed vascular scaffolding may comprise endothelialcells, vascular endothelial cells, pericytes, smooth muscle cells,fibroblasts, endothelial progenitor cells, stem cells, or anycombination thereof.

The first cell group and/or second cell group may comprise endothelialcells, microvascular endothelial cells, pericytes, smooth muscle cells,fibroblasts, endothelial progenitor cells, lymph cells, T cells such ashelper T cells and cytotoxic T cells, B cells, natural killer (NK)cells, reticular cells, hepatocytes, or any combination thereof. Thefirst cell group and/or second cell group may comprise exocrinesecretory epithelial cells, hormone-secreting cells, epithelial cells,nerve cells, adipocytes, kidney cells, pancreatic cells, pulmonarycells, extracellular matrix cells, muscle cells, blood cells, immunecells, germ cells, interstitial cells, or any combination thereof.

The first cell group and/or second cell group may comprise exocrinesecretory epithelial cells including but not limited to salivary glandmucous cells, mammary gland cells, sweat gland cells such as eccrinesweat gland cell and apocrine sweat gland cell, sebaceous gland cells,type II pneumocytes, or any combination thereof.

The first cell group and/or second cell group may comprisehormone-secreting cells including but not limited to anterior pituitarycells, intermediate pituitary cells, magnocellular neurosecretory cells,gut tract cells, respiratory tract cells, thyroid gland cells,parathyroid gland cells, adrenal gland cells, Leydig cells, thecainterna cells, corpus luteum cells, juxtaglomerular cells, macula densacells, peripolar cells, mesangial cells, pancreatic islet cells such asalpha cells, beta cells, delta cells, PP cells, and epsilon cells, orany combination thereof.

The first cell group and/or second cell group may comprise epithelialcells including but not limited to keratinizing epithelial cells such askeratinocytes, basal cells, and hair shaft cells, stratified barrierepithelial cells such as surface epithelial cells of stratified squamousepithelium, basal cells of epithelia, and urinary epithelium cells, orany combination thereof.

The first cell group and/or second cell group may comprise nerve cellsor neurons including but not limited to sensory transducer cells,autonomic neuron cells, peripheral neuron supporting cells, centralnervous system neurons such as interneurons, spindle neurons, pyramidalcells, stellate cells, astrocytes, oligodendrocytes, ependymal cells,glial cells, or any combination thereof.

The first cell group and/or second cell group may comprise kidney cellsincluding but not limited to, parietal cells, podocytes, mesangialcells, distal tubule cells, proximal tubule cells, Loop of Henle thinsegment cells, collecting duct cells, interstitial kidney cells, or anycombination thereof.

The first cell group and/or second cell group may comprise pulmonarycells including, but not limited to type I pneumocyte, alveolar cells,capillary endothelial cells, alveolar macrophages, bronchial epithelialcells, bronchial smooth muscle cells, tracheal epithelial cells, smallairway epithelial cells, or any combination thereof.

The first cell group and/or second cell group may comprise extracellularmatrix cells including, but not limited to epithelial cells,fibroblasts, pericytes, chondrocytes, osteoblasts, osteocytes,osteoprogenitor cells, stellate cells, hepatic stellate cells, or anycombination thereof.

The first cell group and/or second cell group may comprise muscle cellsincluding, but not limited to skeletal muscle cells, cardiomyocytes,Purkinje fiber cells, smooth muscle cells, myoepithelial cells, or anycombination thereof.

The first cell group and/or second cell group may comprise blood cellsand/or immune cells including, but not limited to erythrocytes,megakaryocytes, monocytes, macrophages, osteoclasts, dendritic cells,microglial cells, neutrophils, eosinophils, basophils, mast cells,helper T cells, suppressor T cells, cytotoxic T cells, natural killer Tcells, B cells, natural killer (NK) cells, reticulocytes, or anycombination thereof.

FIG. 2C illustrates delivery of pulses of the multi-photon laser beam120 to the media 126 according to the CAD model of the remaining tissue.Thus, additional portions of the media 126 may polymerize, cross-link orreact to form cell-containing structures 132 around the existingcell-containing scaffolding 128 (no longer visible) without damaging orimpacting the existing vascular scaffolding 128. The steps of drainingthe media 126, refilling with new media 126 and delivering laser energymay be repeated any number of times to create the desired complextissue.

FIG. 2D illustrates an embodiment wherein the cell-containingscaffolding 128 may be printed along the bottom of the media chamber 122containing media 126. Thus, the scaffolding 128 may not be free standingor free floating. The multi-channel input may reduce shear forcesassociated with bulk flow from one direction, uneven washing of finestructures as bulk flow may not wash unwanted cells from small features,and uneven distribution of new cell containing media as it is cycledinto the tissue printing chamber. The multiple inputs may come from thetop, bottom, sides or all three simultaneously. Multiple inputs areparticularly desired for tissue printing because cell-containingstructures are relatively fragile and potentially disrupted by theapplication of fluid forces associated with media exchange through thechamber. FIG. 2D shows that the tissues may be printed above the bottomplate of the media chamber. In some embodiments, the cells and tissuemay be printed flush against the bottom of the media chamber.Additionally, this design may allow for easy transport of printedtissues and positioning under a laser print head (focusing objective)and is a closed system that may allow for media exchange and printing tooccur without exposure to room air. This may be desired as exposure toroom air can introduce infectious agents into the cell culture mediawhich may disrupt or completely destroy the development of usefultissues.

Laser Printing Systems

In an aspect, the present disclosure provides systems for printing athree-dimensional (3D) biological material. The x, y, and z dimensionsmay be simultaneously accessed by the systems provided herein. A systemfor printing a 3D biological material may comprise a media chamberconfigured to contain a medium comprising a plurality of cellscomprising cells and one or more polymer precursors. The plurality ofcells may comprise cells of at least one type. The plurality of cellsmay comprise cells of at least two different types. The system maycomprise at least one energy source configured to direct at least oneenergy beam to the media chamber. The system may comprise at least oneenergy source configured to direct at least one energy beam to the mediachamber and/or to the cell-containing chamber. The system may compriseone or more computer processors operatively coupled to the at least oneenergy source, wherein the one or more computer processors may beindividually or collectively programmed to: receive computerinstructions for printing the 3D biological material from computermemory; and direct the at least one energy source to direct the at leastone energy beam to the medium in the media chamber along at least oneenergy beam path in accordance with the computer instructions, tosubject at least a portion of the polymer precursors to form at least aportion of the 3D biological material.

In another aspect, the present disclosure provides an additional systemfor printing a 3D biological material, comprising a media chamberconfigured to contain a medium comprising a plurality of cells and aplurality of polymer precursors. The system may comprise at least oneenergy source configured to direct at least one energy beam to the mediachamber. In addition, the system may comprise one or more computerprocessors that may be operatively coupled to the at least one energysource. The one or more computer processors may be individually orcollectively programmed to: (i) receive computer instructions forprinting the 3D biological material from computer memory; (ii) directthe at least one energy source to direct the at least one energy beam tothe medium in the media chamber along at least one energy beam path inaccordance with the computer instructions, to subject at least a portionof the polymer precursors to form at least a portion of the 3Dbiological material; and (iii) direct the at least one energy source todirect the at least one energy beam to a second medium in the mediachamber along at least one energy beam path in accordance with thecomputer instructions, to subject at least a portion of the secondmedium in the media chamber to form at least a second portion of the 3Dbiological material, wherein the second medium comprises a secondplurality of cells and a second polymeric precursor, wherein the secondplurality of cells is of a different type than the first plurality ofcells.

The one or more computer processors are individually or collectivelyprogrammed to generate a point-cloud representation or lines-basedrepresentation of the 3D biological material in computer memory, and usethe point-cloud representation or lines-based representation to generatethe computer instructions for printing the 3D biological material incomputer memory. The one or more computer processors may be individuallyor collectively programmed to direct the at least one energy source todirect the at least one energy beam along one or more additional energybeam paths to form at least another portion of the 3D biologicalmaterial.

The system may comprise one or more computer processors operativelycoupled to at least one energy source and/or to at least one lightpatterning element. The point-cloud representation or the lines-basedrepresentation of the computer model may be a holographic point-cloudrepresentation or a holographic lines-based representation. The one ormore computer processors may be individually or collectively programmedto use the light patterning element to re-project the holographic imageas illuminated by the at least one energy source.

In some cases, one or more computer processors may be individually orcollectively programmed to convert the point-cloud representation orlines-based representation into an image. The one or more computerprocessors may be individually or collectively programmed to project theimage in a holographic manner. The one or more computer processors maybe individually or collectively programmed to project the image as ahologram. The one or more computer processors may be individually orcollectively programmed to project the image as partial hologram. Insome cases, one or more computer processors may be individually orcollectively programmed to convert the point-cloud representation orlines-based representation of a complete image set into a series ofholographic images via an algorithmic transformation. This transformedimage set may then be projected in sequence by a light patterningelement, such as a spatial light modulator (SLM) or digital mirrordevice (DMD), through the system, recreating the projected image withinthe printing chamber with the projected light that is distributed in 2Dand or 3D simultaneously. An expanded or widened laser beam may beprojected onto the SLMs and/or DMDs, which serve as projection systemsfor the holographic image. In some cases, one or more computerprocessors may be individually or collectively programmed to project theimage in a holographic manner. In some cases, one or more computerprocessors may be individually or collectively programmed to project theimages all at once or played in series as a video to form a larger 3Dstructure in a holographic manner.

Holography is a technique that projects a multi-dimensional (e.g. 2Dand/or 3D) holographic image or a hologram. When a laser that canphoto-polymerize a medium is projected as a hologram, the laser mayphotopolymerize, solidify, cross-link, bond, harden, and/or change aphysical property of the medium along the projected laser light path;thus, the laser may allow for the printing of 3D structures. Holographymay require a light source, such as a laser light or coherent lightsource, to create the holographic image. The holographic image may beconstant over time or varied with time (e.g., a holographic video).Furthermore, holography may require a shutter to open or move the laserlight path, a beam splitter to split the laser light into separatepaths, mirrors to direct the laser light paths, a diverging lens toexpand the beam, and additional patterning or light directing elements.

A holographic image of an object may be created by expanding the laserbeam with a diverging lens and directing the expanded laser beam ontothe hologram and/or onto at least one pattern forming element, such as,for example a spatial light modulator or SLM. The pattern formingelement may encode a pattern comprising the holographic image into alaser beam path. The pattern forming element may encode a patterncomprising a partial hologram into a laser beam path. Next, the patternmay be directed towards and focused in the medium chamber containing theprinting materials (i.e., the medium comprising the plurality of cellsand polymeric precursors), where it may excite a light-reactivephotoinitiator found in the printing materials (i.e., in the medium).Next, the excitation of the light-reactive photoinitiator may lead tothe photopolymerization of the polymeric-based printing materials andforms a structure in the desired pattern (i.e., holographic image). Insome cases, one or more computer processors may be individually orcollectively programmed to project the holographic image by directing anenergy source along distinct energy beam paths.

In some cases, at least one energy source may be a plurality of energysources. The plurality of energy sources may direct a plurality of theat least one energy beam. The energy source may be a laser. In someexamples, the laser may be a fiber laser. For example, a fiber laser maybe a laser with an active gain medium that includes an optical fiberdoped with rare-earth elements, such as, for example, erbium, ytterbium,neodymium, dysprosium, praseodymium, thulium and/or holmium. The energysource may be a short-pulsed laser. The energy source may be afemto-second pulsed laser. The femtosecond pulsed laser may have a pulsewidth less than or equal to about 500 femtoseconds (fs), 250, 240, 230,220, 210, 200, 150, 100, 50 fs, 40 fs, 30 fs, 20 fs, 10 fs, 9 fs, 8 fs,7 fs, 6 fs, 5 fs, 4 fs, 3 fs, 2 fs, 1 fs, or less. The femtosecondpulsed laser may be, for example, a titanium:sapphire (Ti:Sa) laser. Theat least one energy source may be derived from a coherent light source.

The coherent light source may provide light with a wavelength from about300 nanometers (nm) to about 5 millimeters (mm). The coherent lightsource may comprise a wavelength from about 350 nm to about 1800 nm, orabout 1800 nm to about 5 mm. The coherent light source may provide lightwith a wavelength of at least about 300 nm, 400 nm, 500 nm, 600 nm, 700nm, 800 nm, 900 nm, 1 mm, 1.1 mm, 1.2, mm, 1.3 mm, 1.4 mm, 1.5 mm, 1.6mm, 1.7 mm, 1.8 mm, 1.9 mm, 2 mm, 3 mm, 4 mm, 5 mm, or greater.

The computer processors may be individually or collectively programmedto direct the at least one energy source to direct the at least oneenergy beam along one or more additional energy beam paths to form atleast another portion of the 3D biological material. The one or moreadditional energy beam paths may be along an x axis, an x and y plane,or the x, y, and z planes. The one or more additional energy beam pathsmay be along an x axis. The one or more additional energy beam paths maybe along a y axis. The one or more additional energy beam paths may bealong a z axis. The energy beam path may converge with one or more otherbeams on the same axis. The one or more additional energy beam paths maybe in the x and y plane. The one or more additional energy beam pathsmay be in the x and z plane. The one or more additional energy beampaths may be in the y and z plane. The one or more additional energybeam paths may be in the x, y, and z planes.

The system may further comprise at least one objective lens fordirecting the at least one energy beam to the medium in the mediachamber. In some instances, at least one objective lens may comprise awater-immersion objective lens. In some instances, at least oneobjective lens may comprise a water-immersion objective lens. In someinstances, at least one objective lens may comprise a water dippingobjective lens. In some instances, at least one objective lens maycomprise an oil immersion objective lens. In some instances, at leastone objective lens may comprise an achromatic objective lens, asemi-apochromatic objective lens, a plans objective lens, an immersionobjective lens, a Huygens objective lens, a Ramsden objective lens, aperiplan objective lens, a compensation objective lens, a wide-fieldobjective lens, a super-field objective lens, a condenser objectivelens, or any combination thereof. Non-limiting examples of a condenserobjective lens may include an Abbe condenser, an achromatic condenser,and a universal condenser.

The one or more computer processors may be individually or collectivelyprogrammed to receive images of the edges of the 3D biological material.The one or more computer processors may be individually or collectivelyprogrammed to receive images of the exterior surfaces of the 3Dbiological material. The one or more computer processors may beindividually or collectively programmed to receive images of theinterior surfaces of the 3D biological material. The one or morecomputer processors may be individually or collectively programmed toreceive images of the interior of the 3D biological material.

The one or more computer processors may be individually or collectivelyprogrammed to direct linking of the 3D biological material with othertissue, which linking may be in accordance with the computerinstructions. The one or more computer processors may be individually orcollectively programmed to directly link, merge, bond, or weld 3Dprinted material with already printed structures, where linking is inaccordance with the computer model. In some cases, linking of the 3Dbiological material with other tissue may involve chemicalcross-linking, mechanical linking, and/or cohesively coupling.

In another aspect, the system may comprise a media chamber configured tocontain a medium comprising a plurality of cells and a plurality ofpolymer precursors. The system may comprise at least one energy sourceconfigured to direct at least one energy beam to the media chamber. Thesystem may comprise one or more computer processors operatively coupledto at least one energy source, wherein the one or more computerprocessors are individually or collectively programmed to: receive acomputer model of the 3D biological material in computer memory;generate a point-cloud representation or lines-based representation ofthe computer model of the 3D biological material in computer memory;direct the at least one energy source to direct the at least one energybeam to the medium in the media chamber along at least one energy beampath in accordance with the computer model of the 3D biologicalmaterial, to subject at least a portion of the polymer precursors toform at least a portion of the 3D biological material; and direct the atleast one energy source to direct the at least one energy beam to asecond medium in the media chamber along at least one energy beam pathin accordance with the computer model of the 3D biological material, tosubject at least a portion of the second medium in the media chamber toform at least a second portion of the 3D biological material, whereinthe second medium comprises a second plurality of cells and a secondpolymeric precursor, wherein the second plurality of cells is of adifferent type than the first plurality of cells.

In laser printing of cellular structures, rapid three-dimensionalstructure generation using minimally toxic laser excitation is criticalfor maintaining cell viability and in the case of functional tissueprinting, necessary for large-format, high resolution, multicellulartissue generation. Other methods of two-photon printing may rely uponraster-scanning of two-photon excitation in a two-dimensional plane (x,y) (e.g., selective laser sintering), while moving the microscope orstage in the z direction to create a three-dimensional structure. Thistechnique may be prohibitively slow for large format multicellulartissue printing such that cell viability may be unlikely to bemaintained during printing of complex structures. Certain hydrogels withhigh rates of polymerization may also be utilized for two-dimensionalprojection of tissue sheets that are timed such that one slice of astructure is projected with each step in an x, y, or z plane.Additionally, mixed plane angles representing a sheet or comprising anorthogonal slice may also be utilized. In the case of rapidlypolymerizing hydrogels, these projections may work in time-scales thatare compatible with tissue printing whereas laser sintering or rasterscanning (e.g. layer-by-layer deposition) may be prohibitively slow forbuilding a complex structure.

The laser printing system 110 of the present disclosure may be equippedwith an objective lens 124 that may allow for focusing of thethree-dimensional or two-dimensional holographic projection in thelateral and axial planes for rapid creation of cell containingstructures. The objective lens 124 may be a water-immersion objectivelens, an air objective lens, or an oil-immersion objective lens. In somecases, the laser printing system 110 may include a laser system 116having multiple laser lines and may be capable of three-dimensionalholographic projection of images for photolithography via holographicprojection into cell containing media.

FIG. 3A illustrates an embodiment of a laser system 116 having a firstmulti-photon laser source 140 a. Here, the laser line one, multi-photonlaser beam may be reflected by a spatial light modulator (SLM) with avideo rate or faster re-fresh rate for image projection, to allow forrapid changes in the three-dimensional structure being projected.

In some cases, spatial light modulators (SLMs) may be used to print a 3Dbiological material. In some cases, the method presented herein maycomprise receiving a computer model of the 3D biological material incomputer memory and further processing the computer model such that thecomputer model is “sliced” into layers, creating a two-dimensional (2D)image of each layer. The computer model may be a computer-aided design(CAD) model. The system disclosed herein may comprise at least onecomputer processor which may be individually or collectively programmedto calculate a laser scan path based on the “sliced” computer model,which determines the boundary contours and/or fill sequences of the 3Dbiological material to be printed. Holographic 3D printing may be usedwith one or more polymer precursors described herein. SLM may be usedwith two or more polymer precursors described herein.

A spatial light modulator (SLM) is an electrically programmable devicethat can modulate amplitude, phase, polarization, propagation direction,intensity or any combination thereof of light waves in space and timeaccording to a fixed spatial (i.e., pixel) pattern. The SLM may be basedon translucent, e.g. liquid crystal display (LCD) microdisplays. The SLMmay be based on reflective, e.g. liquid crystal on silicon (LCOS)microdisplays. The SLM may be a microchannel spatial light modulator(MSLM), a parallel-aligned nematic liquid crystal spatial lightmodulator (PAL-SLM), a programmable phase modulator (PPM), a phasespatial light modulator (LCOS-SLM), or any combination thereof. AnLCOS-SLM may comprise a chip that includes a liquid crystal layerarranged on top of a silicon substrate. A circuit may be built on thechip's silicon substrate by using semiconductor technology. A top layerof the LCOS-SLM chip may contain aluminum electrodes that are able tocontrol their voltage potential independently. A glass substrate may beplaced on the silicon substrate while keeping a constant gap, which isfilled by the liquid crystal material. The liquid crystal molecules maybe aligned in parallel by the alignment control technology provided inthe silicon and glass substrates. The electric field across this liquidcrystal layer can be controlled pixel by pixel. The phase of light canbe modulated by controlling the electric field; a change in the electricfield may cause the liquid crystal molecules to tilt accordingly. Whenthe liquid crystal molecules tilt, the liquid crystal refractive indexesmay change further changing the optical path length and thus, causing aphase difference.

An SLM may be used to print the 3D biological material. A liquid crystalon silicon (LCOS)-SLM may be used to print the 3D biological material. Aliquid crystal SLM may be used to print the 3D biological material. TheSLM may be used to project a point-cloud representation or a lines-basedrepresentation of a computer model of the 3D biological material. Themethods disclosed herein may comprise converting the point-cloudrepresentation or lines-based representation into a holographic image.The SLM may be used to project the holographic image of the computermodel of the 3D biological material. The SLM may be used to modulate thephase of light of a point-cloud representation or a lines-basedrepresentation of a computer model of the 3D biological material. TheSLM may be used to modulate the phase of light of the holographic imageof the computer model of the 3D biological material.

Projection of multi-photon excitation in three dimensions can also beachieved with the use of a dual digital micromirror device (DMD) systemalone or in combination with a spatial light modulator (SLM). A pair ofDMDs may be used with a pair of SLMs to print a 3D material using themethods described herein. At least one SLM and at least one DMD may beused to print a 3D material using the methods described herein. A pairof SLMs may be used to print a 3D material using the methods describedherein. A pair of DMDs may be used to print a 3D material using themethods described herein. At least one SLM may be used to print a 3Dmaterial using the methods described herein. At least one DMD may beused to print a 3D material using the methods described herein. A DMD isan electrical input, optical output micro-electrical-mechanical system(MEMS) that allows for high speed, efficient, and reliable spatial lightmodulation. A DMD may comprise a plurality of microscopic mirrors(usually in the order of hundreds of thousands or millions) arranged ina rectangular array. Each microscopic mirror in a DMD may correspond toa pixel of the image to be displayed and can be rotated about e.g.10-12° to an “on” or “off” state. In the “on” state, light from aprojector bulb can be reflected into the microscopic mirror making itscorresponding pixel appear bright on a screen. In the “off” state, thelight can be directed elsewhere (usually onto a heatsink), making themicroscopic mirror's corresponding pixel appear dark. The microscopicmirrors in a DMD may be composed of highly reflective aluminum and theirlength across is approximately 16 micrometers (μm). Each microscopicmirror may be built on top of an associated semiconductor memory celland mounted onto a yoke which in turn is connected to a pair of supportposts via torsion hinges. The degree of motion of each microscopicmirror may be controlled by loading each underlying semiconductor memorycell with a “1” or a “0.” Next, a voltage is applied, which may causeeach microscopic mirror to be electrostatically deflected about thetorsion hinge to the associated +/− degree state via electrostaticattraction.

With reference to FIGS. 3A-3C, the addition of an optional beam expanderfollowed by a Bessel beam generating lens that is either a fixed axiconor a tunable acoustic gradient (TAG) lens may be added to alter theproperties of the laser to achieve higher resolution and greater tissueprinting depth, particularly in turbid solutions. The laser line, whichmay include the optional beam expander and/or Bessel beam generatinglens, is directed with fast switch mirrors to distinct projectionsystems that have material advantages in the formation of specificstructures associated with tissue printing. In some cases, a highresolution DMD mirror in conjunction with an SLM system may achievehigher axial resolution than is capable with two SLM systems. Finally, alaser line may be used with a single DMD or SLM system in conjunctionwith a mirror to allow for scan-less projection of a two-dimensionalimage in any of the axial planes. A 3D projection pattern may also beraster-scanned across a larger field of view by scan mirrors where inlaser emission patterns, wavelength, and or power is controlled to matchthe raster scan speed such that a cohesive and complex structure may bedeposited. Within the system containing more than one laser line theconfigurations may be any combination of dual SLM, dual DMD, single SLM,single DMD or simple planar scanning.

In some cases, one or more light paths, such as the ones shown in FIGS.3A-3C, may be used independently or in concert. The lenses, gratings,and mirrors that focus and distribute the light or energy beam withinthe optical path may be placed between the primary, wave-front shapingelements necessary to distribute the light through key elements ormodulate incoming light in the case of a grating, as described in FIG.3A. At least one grating or mirror may be placed between wave-frontshaping elements “F” (i.e., between an SLM, a DMD, and/or a TAG lens)for the purpose of focusing, distributing, or clipping the input laserlight. The optical wave-front shaping device F may comprise an SLM, anLCOS-SLM, a DMD, a TAG lens, or any combination thereof.

In some cases, a DMD may be used to print a 3D biological material. TheDMD may be used to project a point-cloud representation or a lines-basedrepresentation of a computer model of the 3D biological material. Themethods disclosed herein may comprise converting the point-cloudrepresentation or lines-based representation into a holographic image.The DMD may be used to project the holographic image of the computermodel of the 3D biological material. The DMD may be used to print the 3Dbiological material.

In some cases, a combination of at least one SLM and at least one DMDmay be used in the methods disclosed herein to print the 3D biologicalmaterial. The combination of at least one SLM and at least one DMD maybe arranged in series. The combination of at least one SLM and at leastone DMD may be arranged in parallel. The combination of any number ofSLMs and any number of DMDs may be arranged in series when used to printthe 3D biological material. The combination of any number of SLMs andany number of DMDs may be arranged in parallel when used to print the 3Dbiological material.

The combination of at least two SLMs and at least one DMD may be used toprint the 3D biological material. The combination of at least three SLMsand at least one DMD may be used to print the 3D biological material.The combination of at least four SLMs and at least one DMD may be usedto print the 3D biological material. The combination of at least fiveSLMs and at least one DMD may be used to print the 3D biologicalmaterial. The combination of at least ten SLMs and at least one DMD maybe used to print the 3D biological material. The combination of at leasttwenty SLMs and at least one DMD may be used to print the 3D biologicalmaterial.

The combination of at least one SLM and at least two DMDs may be used toprint the 3D biological material. The combination of at least one SLMand at least three DMDs may be used to print the 3D biological material.The combination of at least one SLM and at least four DMDs may be usedto print the 3D biological material. The combination of at least one SLMand at least five DMDs may be used to print the 3D biological material.The combination of at least one SLM and at least ten DMDs may be used toprint the 3D biological material. The combination of at least one SLMand at least twenty DMDs may be used to print the 3D biologicalmaterial.

The combination of at least two SLMs and at least two DMDs may be usedto print the 3D biological material. The combination of at least threeSLMs and at least three DMDs may be used to print the 3D biologicalmaterial. The combination of at least four SLMs and at least four DMDsmay be used to print the 3D biological material. The combination of atleast five SLMs and at least five DMDs may be used to print the 3Dbiological material. The combination of at least ten SLMs and at leastten DMDs may be used to print the 3D biological material. Thecombination of at least twenty SLMs and at least twenty DMDs may be usedto print the 3D biological material.

A liquid crystal SLM may be used to print the 3D biological material. Aplurality of SLMs may be used to print the 3D biological material. Theplurality of SLMs can be arranged in series. The plurality of SLMs canbe arranged in parallel. At least one or more SLMs may be used to printthe 3D biological material. At least two or more SLMs may be used toprint the 3D biological material. At least three or more SLMs may beused to print the 3D biological material. At least four or more SLMs maybe used to print the 3D biological material. At least five or more SLMsmay be used to print the 3D biological material. At least ten or moreSLMs may be used to print the 3D biological material. At least twenty ormore SLMs may be used to print the 3D biological material. At least oneto about fifty or more SLMs may be used to print the 3D biologicalmaterial. At least one to about twenty or more SLMs may be used to printthe 3D biological material. At least one to about fifteen or more SLMsmay be used to print the 3D biological material. At least one to aboutten or more SLMs may be used to print the 3D biological material. Atleast one to about five or more SLMs may be used to print the 3Dbiological material.

A plurality of DMDs may be used to print the 3D biological material. Theplurality of DMDs can be arranged in series. The plurality of DMDs canbe arranged in parallel. At least one or more DMDs may be used to printthe 3D biological material. At least two or more DMDs may be used toprint the 3D biological material. At least three or more DMDs may beused to print the 3D biological material. At least four or more DMDs maybe used to print the 3D biological material. At least five or more DMDsmay be used to print the 3D biological material. At least ten or moreDMDs may be used to print the 3D biological material. At least twenty ormore DMDs may be used to print the 3D biological material. At least oneto about fifty or more DMDs may be used to print the 3D biologicalmaterial. At least one to about twenty or more DMDs may be used to printthe 3D biological material. At least one to about fifteen or more DMDsmay be used to print the 3D biological material. At least one to aboutten or more DMDs may be used to print the 3D biological material. Atleast one to about five or more DMDs may be used to print the 3Dbiological material.

In this design, SLM may refer to liquid crystal SLM and the function ofthe DMD may be similar to the SLM. These lasers may be controlled by oneor more computer inputs to address location and print timing of multiplelaser lines. An example overall design for the light path, includingoptional in-series excitations paths is illustrated in FIG. 3A alongwith further description of the elements provided in Table 1. Because ofthe extensive pulse-width between packets of two photon excitationlight, any combination of these laser lines, which may benon-interfering, may be used simultaneously for printing and printingwith simultaneous imaging. This may permit the interference between thebeams to be substantially low such that the beams to not intersect.Therefore, the use of multiple laser lines with minimal to nointerference is possible as illustrated in FIGS. 3B-3C along withfurther description of the elements also provided in Table 1. The groupdelay dispersion optical element in this configuration may be used todisperse two-photon packets such that the peak power output does notdamage a fiber optic cable if one is to be used in certainconfigurations. In addition, group delay dispersion can concentratephotons into shorter pulse-widths such that more energy is imparted atthe focal point or in the projected image allowing for more rapidprinting.

Two photon excitation pulses may be temporally controlled such thatexcitation at a single spot occurs with pulses that are femto- tonanosecond range in length (dependent on laser tuning) while the timingbetween these photon packets is three to six orders of magnitude longerthan the pulse width. This may allow for minimal cross-path interferenceof laser excitations making use of multiple lasers for simultaneousprinting possible when using multiple laser lines in series. An exampleof multiple laser projections at three different theoretical wavelengthsfor the purpose of structure deposition is presented in FIG. 3B.Multi-photon lasers are tunable; thus, they may allow for a range ofwavelengths to be selected. This is advantageous in tissue printingwherein different photoinitiators for polymerization that respond todifferent wavelengths may be used in combination or in series to preventunwanted polymerization of left-over materials. Therefore, each of theselaser lines may be tuned to a different multi-photon output wavelength,may have different peak power output, and may project a differentelement of the CAD image that comprises the tissue structure.

TABLE 1 Element descriptions for FIGS. 3A-3C Element Label Description140a-c Laser source. A first laser source 140a, a second laser source140b, and a third laser source 140c may be a tunable multi-photon(femto-second pulsed) laser of a given power (e.g. between 1 and 50watts and 640 to 1500 nm wavelength output). Femto- second laser sourcesmay be tunable by computer software interaction and thus may be set tovarious wavelengths before or during the printing process to producedifferent excitation wavelengths. Optionally, the systems disclosedherein may have a pump laser system. A Mirror. A mirror with or withoutan infrared (IR) specific coating to improve reflectance. IR specificcoating examples may include protected gold or protected silver basedcoatings. As shown in FIG. 3A, grating and/or mirrors may be addedbetween elements “F” (i.e., between DMDs, SLMs, or TAG lenses). B Beamexpander. An optional beam expander to expand the area of the laserpulse prior to projection by the DMD or SLM systems. C Axicon or TAGlens. In some tissue printing applications, the use of a Bessel beam mayallow for improved or even power output at greater depths in hydrogels,media, or already printed structures. To produce a Bessel beam, anaxicon which produces a fixed Bessel beam or tunable acoustic gradientlens (TAG), may produce a Bessel beam that is tunable and can be alteredby altering an electric signal input. In the instance that a TAG lens isused, the input signal may be controlled by integrated computersoftware. D Dispersion compensation unit. The purpose of the dispersioncompensation unit in this design is to concentrate emitted two-photonpackets such that the peak power output is higher at the excitationpoint. This allows for improved polymerization as a result of improvedpeak power output at a specific wavelength. E Beam Dump. Beam dumpallows for collection of stray laser light. F DMD, SLM, or TAG lens. Inthis example design, a DMD or SLM may be used to create an x, y plane ofprojection with a specific pattern of light that may be used topolymerize the monomers into structures or nets that contain cells. Theaddition of the second DMD or SLM may allow for projection of the x, yplane in the z or axial direction for three-dimensional holographicprojection of the multiphoton excitation into the print vessel. This mayallow for polymerization of the structures in three dimensions whereinall x, y, and z dimension features are deposited at the same time. EachDMD or SLM may be controlled by computer input and may be directed toproject a specific CAD image or portion of a CAD image. Having the SLMor DMDs in series may allow for images to be projected simultaneously indifferent wavelengths of light in the case of multiple laser excitationsources (such as illustrated in FIG. 3B) or in the case of multiplerepeating pattern projection SLMs or DMDs can be used to projectdifferent aspects of the same tissue without needing to switch thecomputer input, instead mirrors can be used to re-direct or turn ‘off’or ‘on’ a particular light path and produce a given fixed structureassociated with laser light paths 1, 2, 3, or 4. In cases where theBessel beam is removed (element C), this may allow for different axialaccuracies in printing a particular given structure. Therefore, certainelements of tissue structure may be better printed by different lightpaths. Rapid switching between laser light paths can allow for printingand polymerization to continue while an SLM or DMD series isre-programed for projection of the next tissue structure in a givenseries of printing steps. In some cases, element “F” can represent a TAGlens. The TAG lens as used as element “F” can manipulate light. The TAGlens as used as element “F” can holographically distribute light. GMovable mirror. A mirror with or without an IR specific coating toimprove reflectance. IR specific coating examples may include protectedgold or protected silver based coatings. These mirrors can be moveableand can be adjusted to be in an ‘on’ or ‘off’ state to redirect thelaser light path through the printing system as desired. Control ofmirror positioning may be dictated by computer software. H Beamcombiner. Beam combiner allowing for multiple light paths to berecombined for simultaneous printing at different wavelengths. In FIG.3B these may also be movable mirrors (G) that can allow for the samewavelengths to be printed with timed on/off states of the mirrors G. ILight path to the optics housing. J Band pass filter. The purpose of anoptional band-pass filter may be to select a specific wavelength to beused in materials polymerization. Multi-photon excitation may have anemission spread that can span several tens of nanometers potentiallyleading to overlap in absorption and thus polymerization of materialswith otherwise distinct absorption peaks. By selecting for specificwavelengths using a band pass filter the wavelength leading topolymerization may be fine-tuned to prevent undesirable cross-overeffects when two different monomers with different responsiveness usedin the same formulation. K Scan head. Two mirrors that representoptional laser light scanning or sintering in the x, y plane. Thesemirrors may vibrate at a given frequency, for example 20 kHz, one in thex-direction reflecting to the next mirror which may scan in the ydirection. This scanning may create a plane of light that can be used toimage tissues or polymerized units before, after, and during thepolymerization process. This is possible as collagen and many otherordered structures can emit light via a non-linear process call secondharmonic generation when polymerized but not when in a monomeric state.Therefore, using an additional excitation source tuned to a wavelengththat may allow for second harmonic generation and imaging while notpolymerizing the biomaterials can be useful for monitoring the printingprocess. L Long pass Mirror: A long pass mirror may allow multi-photonexcitation from light path number 4 to pass through while reflecting anyemission from a sample while in imaging mode (requires engagement oflaser light path 4) to the series of photomultiplier tubes (PMT) Mdetectors and long pass or band pass mirrors of various wavelengths thatmay allow for specific emission wavelengths to be reflected into thePMTs for image collection via personal computer (PC) (i.e., computerprocessor) and appropriate imaging processing software. M Photomultiplier tubes. PMTs may be used in collection of images inmicroscopy. N Objective. This objective may serve the purpose ofconcentrating the multiphoton excitation such that polymerization ofmonomers to match the projected image may take place. O Movable longpass mirror. In instances where imaging may be performed with light path#4 the mirror O may be moved via software control to allow for laserlight path 4 to enter the objective (N). In some incarnations light path4 may be tuned to a distinct wavelength from laser light paths 1, 2, or3 allowing for a long or short pass mirror or beam combiner to be usedin place of O. 1 Laser light path 1 may be used to by-pass the beamexpansion or beam expansion plus Bessel beam lens combination in favorof direct transmittance into the SLM/DMD series or individual SLM orDMD. Laser line one may also be redirected into laser line 5 whichcreates a single two photon pinpoint excitation, which may be used inoptics housing alignment or raster scanning of a sample for imagingpurposes. 2 & 5 Laser light path 2 may be transmitted through anoptional beam expander and optional Bessel beam creating lens (axicon orTAG lens) then a single SLM or DMD and may also be re-directed to laserlight path 5. 3 & 4 Laser light paths 3 and 4 may be passed through anoptional beam expander and optional Bessel beam creating lens (axicon orTAG lens) followed by a combination of SLM or DMDs in series. Twodistinct laser lines may allow for construction of dual SLM, dual DMD ora combination of the two which can increase flexibility in printingdifferent sizes and types of structures. Furthermore, the laser line canbe flickered between two different structures projected by each seriesto allow for near-simultaneous printing of complex structures that maynot otherwise be achieved with a single DMD or SLM series. At any timethese laser lines may be re-directed to the beam dump E which functionsas a default off state.

FIGS. 4A-4B demonstrates the placement of an optional beam expanderprior to the axicon or tunable acoustic gradient (TAG) lens. This mayallow for generation of a Bessel beam for the purpose of increased depthpenetration in tissues and turbid media during printing without loss offocus fidelity. This feature may improve depth of printing throughturbid media or through already formed tissues without loss of power.

A lens may be used to either widen or pre-focus the laser after the dualSLM or DMD combination. In addition, a laser attenuation device orfiltering wheel that is computer controlled may be added prior tofocusing optics to control the laser power output at the site ofprinting.

FIG. 4C illustrates a laser source A projecting a laser beam onto a beamcollector B. Upon exiting the beam collector B, the laser beam may bedirected to an optical TAG or axicon C and further to a movable, singleSLM or DMD D for 2D x, y sheet projection for collagen net printingaround cells and resultant structures printed with given Z-steps. Thelaser beam may be directed from the SLM or DMD D into a mirror G andthen reflected onto the print head optics H. In this example, atwo-dimensional (2D) projection may be created with a single SLM with az-motor-stepped movement that matches the frame rate of the projection.Two-dimensional video projection of the z-stack slice may be achievedwith a single DMD or a single SLM that is timed with z-movement suchthat each step projects a distinct image printing a 2D image from thetop down. In another embodiment, a complex structure may be projectedfrom the side, bottom up, or a different articulation and slice byslice, 2D projected and printed using either multi-photon or alternativelaser excitation source. The source of CAD images F may be directed fromthe computer E into the system. The system may comprise a motorizedstage I that may match the step rate (millisecond to second) and thestep size of a Z-projection. The step size may be in the order ofmicrons to nanometers. In FIGS. 4C, 1, 2, and 3 illustrate examples ofplanar projection build steps.

FIG. 44 illustrates the optical components and the optical path of anembodiment of the three-dimensional printing system. The opticalcomponents and the optical path shown in FIG. 44 may provide athree-dimensional printing system that may not use temporal focusing.The three-dimensional printing system may comprise an energy source1000. The energy source 1000 may be a coherent light source. The energysource 1000 may be a laser light. The energy source 1000 may be afemto-second pulsed laser light source. The energy source 1000 may be afirst laser source 140 a, a second laser source 140 b, or a third lasersource 140 c. The energy source 1000 may be a multi-photon laser beam120. The energy source 1000 may be a two-photon laser beam. The energysource 1000 may be controlled by a computer system 1101. The energysource 1000 may be tuned by a computer system 1101. The computer system1101 may control and/or set the energy wavelength of the energy source1000 prior to or during the printing process. They computer system 1101may produce different excitation wavelengths by setting the wavelengthof the energy source 1000.

The energy source 1000 may be pulsed. The energy source 1000 may bepulsed at a rate of about 500 kilohertz (kHz). The energy source 1000(e.g., laser) may provide energy (e.g., laser beam) having energypackets with pulsed energies (per packet) from about at least 1 microjoule (μJ) to 1,000,000 μJ. The energy source 1000 (e.g., laser) mayprovide energy (e.g., laser beam) having energy packets with pulsedenergies (per packet) from about at least 1 micro joule (μJ) to 100,000μL or more. The energy source 1000 (e.g., laser) may provide energy(e.g., laser beam) having energy packets with pulsed energies (perpacket) from about at least 1 micro joule (μJ) to 1,000 μL or more. Theenergy source 1000 (e.g., laser) may provide energy (e.g., laser beam)having energy packets with pulsed energies (per packet) from about atleast 1 micro joule (μJ) to 100 μL or more. The energy source 1000(e.g., laser) may provide energy (e.g., laser beam) having energypackets with pulsed energies (per packet) from about at least 10 microjoule (μJ) to 100 μL or more. The energy source 1000 (e.g., laser) mayprovide energy (e.g., laser beam) having energy packets with pulsedenergies (per packet) from about at least 1 micro joule (μJ) to 50 μL ormore. The energy source 1000 (e.g., laser) may provide energy (e.g.,laser beam) having energy packets with pulsed energies (per packet) fromabout at least 1 micro joule (μJ) to 20 μL or more. The energy source1000 (e.g., laser) may provide energy (e.g., laser beam) having energypackets with pulsed energies (per packet) from about at least 1 microjoule (μJ) to 50 μL or more. The energy source 1000 (e.g., laser) mayprovide energy (e.g., laser beam) having energy packets with pulsedenergies (per packet) from about at least 40 micro joule (μJ) to 80 μLor more. The energy source 1000 (e.g., laser) may provide energy (e.g.,laser beam) having energy packets with pulsed energies (per packet) fromabout at least 120 micro joule (μJ) to 160 μL or more.

The energy source 1000 (e.g., laser) may provide energy (e.g., laserbeam) having energy packets with pulsed energies (per packet) of about10 μJ. The energy source 1000 (e.g., laser) may provide energy (e.g.,laser beam) having energy packets with pulsed energies (per packet) ofabout 20 μJ. The energy source 1000 (e.g., laser) may provide energy(e.g., laser beam) having energy packets with pulsed energies (perpacket) of about 30 μJ. The energy source 1000 (e.g., laser) may provideenergy (e.g., laser beam) having energy packets with pulsed energies(per packet) of about 40 μJ. The energy source 1000 (e.g., laser) mayprovide energy (e.g., laser beam) having energy packets with pulsedenergies (per packet) of about 50 μJ. The energy source 1000 (e.g.,laser) may provide energy (e.g., laser beam) having energy packets withpulsed energies (per packet) of about 60 μJ. The energy source 1000(e.g., laser) may provide energy (e.g., laser beam) having energypackets with pulsed energies (per packet) of about 70 μJ. The energysource 1000 (e.g., laser) may provide energy (e.g., laser beam) havingenergy packets with pulsed energies (per packet) of about 80 μJ. Theenergy source 1000 (e.g., laser) may provide energy (e.g., laser beam)having energy packets with pulsed energies (per packet) of about 90 μJ.The energy source 1000 (e.g., laser) may provide energy (e.g., laserbeam) having energy packets with pulsed energies (per packet) of about100 μJ. The energy source 1000 (e.g., laser) may provide energy (e.g.,laser beam) having energy packets with pulsed energies (per packet) ofabout 110 μJ. The energy source 1000 (e.g., laser) may provide energy(e.g., laser beam) having energy packets with pulsed energies (perpacket) of about 120 μJ. The energy source 1000 (e.g., laser) mayprovide energy (e.g., laser beam) having energy packets with pulsedenergies (per packet) of about 130 μJ. The energy source 1000 (e.g.,laser) may provide energy (e.g., laser beam) having energy packets withpulsed energies (per packet) of about 140 μJ. The energy source 1000(e.g., laser) may provide energy (e.g., laser beam) having energypackets with pulsed energies (per packet) of about 150 μJ. The energysource 1000 (e.g., laser) may provide energy (e.g., laser beam) havingenergy packets with pulsed energies (per packet) of about 160 μJ. Theenergy source 1000 (e.g., laser) may provide energy (e.g., laser beam)having energy packets with pulsed energies (per packet) of about 170 μJ.The energy source 1000 (e.g., laser) may provide energy (e.g., laserbeam) having energy packets with pulsed energies (per packet) of about180 μJ. The energy source 1000 (e.g., laser) may provide energy (e.g.,laser beam) having energy packets with pulsed energies (per packet) ofabout 190 μJ. The energy source 1000 (e.g., laser) may provide energy(e.g., laser beam) having energy packets with pulsed energies (perpacket) of about 200 μJ. The energy source 1000 (e.g., laser) mayprovide energy (e.g., laser beam) having energy packets with pulsedenergies (per packet) of about 20,000 μJ. The energy source 1000 (e.g.,laser) may provide energy (e.g., laser beam) having energy packets withpulsed energies (per packet) of about 100,000 μJ. The energy source 1000(e.g., laser) may provide energy (e.g., laser beam) having energypackets with pulsed energies (per packet) of about 1,000,000 μJ.

The energy source 1000 (e.g., laser) may provide an energy beam (e.g.,light beam) having a wavelength from e.g. about at least 300 nm to about5 mm or more. The energy source 1000 (e.g., laser) may provide energy(e.g., laser beam) having a wavelength of about at least 600 to about1500 nm or more. The energy source 1000 (e.g., laser) may provide energy(e.g., laser beam) having a wavelength from about at least 350 nm toabout 1800 nm or more. The energy source 1000 (e.g., laser) may provideenergy (e.g., laser beam) having a wavelength from about at least 1800nm to about 5 mm or more. The energy source 1000 (e.g., laser) mayprovide energy (e.g., laser beam) having a wavelength of about 300 nm.The energy source 1000 (e.g., laser) may provide energy (e.g., laserbeam) having a wavelength of about 400 nm. The energy source 1000 (e.g.,laser) may provide energy (e.g., laser beam) having a wavelength ofabout 600 nm. The energy source 1000 (e.g., laser) may provide energy(e.g., laser beam) having a wavelength of about 700 nm. The energysource 1000 (e.g., laser) may provide energy (e.g., laser beam) having awavelength of about 800 nm. The energy source 1000 (e.g., laser) mayprovide energy (e.g., laser beam) having a wavelength of about 900 nm.The energy source 1000 (e.g., laser) may provide energy (e.g., laserbeam) having a wavelength of about 1000 nm. The energy source 1000(e.g., laser) may provide energy (e.g., laser beam) having a wavelengthof about 1100 nm. The energy source 1000 (e.g., laser) may provideenergy (e.g., laser beam) having a wavelength of about 1200 nm. Theenergy source 1000 (e.g., laser) may provide energy (e.g., laser beam)having a wavelength of about 1300 nm. The energy source 1000 (e.g.,laser) may provide energy (e.g., laser beam) having a wavelength ofabout 1400 nm. The energy source 1000 (e.g., laser) may provide energy(e.g., laser beam) having a wavelength of about 1500 nm. The energysource 1000 (e.g., laser) may provide energy (e.g., laser beam) having awavelength of about 1600 nm. The energy source 1000 (e.g., laser) mayprovide energy (e.g., laser beam) having a wavelength of about 1700 nm.The energy source 1000 (e.g., laser) may provide energy (e.g., laserbeam) having a wavelength of about 1800 nm. The energy source 1000(e.g., laser) may provide energy (e.g., laser beam) having a wavelengthof about 1900 nm. The energy source 1000 (e.g., laser) may provideenergy (e.g., laser beam) having a wavelength of about 2000 nm. Theenergy source 1000 (e.g., laser) may provide energy (e.g., laser beam)having a wavelength of about 3000 nm. The energy source 1000 (e.g.,laser) may provide energy (e.g., laser beam) having a wavelength ofabout 4000 nm. The energy source 1000 (e.g., laser) may provide energy(e.g., laser beam) having a wavelength of about 5000 nm.

As shown in FIG. 44, the energy source 1000 may project a laser beam1002 through a shutter 1004. Once the laser beam 1002 exits the shutter1004, the laser beam 1002 may be directed through a rotating half-waveplate 1006. Rotating half-wave plates may be transparent plates with aspecific amount of birefringence that may be used mostly formanipulating the polarization state of light beams. Rotating half-waveplates may have a slow axis and a fast axis (i.e., two polarizationdirections), which may be both perpendicular to the direction of thelaser beam 1002. The rotating half-wave plate 1006 may alter thepolarization state of the laser beam 1002 such that the difference inphase delay between the two linear polarization directions is π. Thedifference in phase delay may correspond to a propagation phase shiftover a distance of λ/2. Other types of wave plates may be utilized withthe system disclosed herein; for example, a rotating quarter-wave platemay be used. The rotating half-wave plate 1006 may be a true zero-orderwave plate, a low order wave plate, or a multiple-order wave plate. Therotating half-wave plate 1006 may be composed of crystalline quartz(SiO₂), calcite (CaCO₃), magnesium fluoride (MgF₂), sapphire (Al₂O₃),mica, or a birefringent polymer.

The laser beam 1002 may exit the rotating half-wave plate 1006 and maybe directed through a polarizing beam splitter 1008. The polarizing beamsplitter 1008 may split the laser beam 1002 into a first laser beam 1002a and a second laser beam 1002 b. The first laser beam 1002 a may bedirected to a beam dump 1010. The beam dump 1010 is an optical elementthat may be used to absorb stray portions of a laser beam. The beam dump1010 may absorb the first laser beam 1002 a. The first laser beam 1002 amay be a stray laser beam. The beam dump 1010 may absorb the secondlaser beam 1002 b. The second laser beam 1002 b may be a stray laserbeam. The laser beam 1002 may be directed into the beam dump 1010 in itsentirety and thus, may serve as a default “off” state of the printingsystem. The second laser beam 1002 b may be directed to a beam expander1012. The beam expander 1012 may expand the size of the laser beam 1002b. The beam expander 1012 may increase the diameter of the input secondlaser beam 1002 b to a larger diameter of an output, expanded laser beam1054. The beam expander 1012 may be a prismatic beam expander. The beamexpander 1012 may be a telescopic beam expander. The beam expander 1012may be a multi-prism beam expander. The beam expander 1012 may be aGalilean beam expander. The beam expander 1012 may provide a beamexpander power of about 2×, 3×, 5×, 10×, 20×, or 40×. The beam expander1012 may provide a beam expander power ranging from about 2× to about5×. The beam expander 1012 may provide continuous beam expansion betweenabout 2× and about 5×. The beam expander 1012 may provide a beamexpander power ranging from about 5× to about 10×. The beam expander1012 may provide continuous beam expansion between about 5× and about10×. The expanded laser beam 1054 may be collimated upon exiting thebeam expander 1012.

After exiting the beam expander 1012, the expanded laser beam 1054 maybe directed to a first mirror 1014 a, which may re-direct the expandedlaser beam 1054 to a spatial light modulator (SLM) 1016. The SLM 1016may be controlled by a computer system 1101. The SLM 1016 may bedirected to project a specific image or a specific portion of an imageof a material to be printed using the methods and systems disclosedherein. The material to be printed may be a biological material. Thebiological material may be a three-dimensional biological material. Thespecific image or the specific portion of the image may beone-dimensional, two-dimensional, and/or three-dimensional. The SLM 1016may be directed to project at least one image simultaneously indifferent wavelengths of light. The SLM 1016 may be directed to projectdifferent aspects of the material to be printed with the use of mirrorsinstead of with the use of a computer system 1101. In some cases, atleast one mirror may be used to re-direct or turn “off” or “on” aparticular light path or laser beam in order to print different aspectsor portions of the material to be printed.

After exiting the SLM 1016, the expanded laser beam 1054 may be directedto an f1 lens 1018. The f1 lens 1018 may be a focusing lens. Afterexiting the f1 lens 1018, the expanded laser beam 1054 may be directedto blocking element 1020. The blocking element 1020 may be immovable.The blocking element 1020 may suppress illumination from a zero-orderspot. A zero-order may be a part of the energy from the expanded laserbeam 1054 that is not diffracted and behaves according to the laws orreflection and refraction. After exiting the blocking element 1020, theexpanded energy beam 1054 may be directed through an f2 lens 1022. Thef2 lens may be a focusing lens.

After exiting the f2 lens 1022, the expanded laser beam 1054 may bedirected onto a second mirror 1014 b and may be subsequently directedonto a third mirror 1014 c. The third mirror 1014 c may re-direct theexpanded laser beam 1054 through a long pass dichroic mirror 1024. Thefirst mirror 1014 a, the second mirror 1014 b, and/or the third mirror1014 c may comprise an infrared (IR) coating to improve reflectance. Thefirst mirror 1014 a, the second mirror 1014 b, and/or the third mirror1014 c may not comprise an infrared (IR) coating. Non-limiting examplesof IR coatings include protected gold-based coatings and protectedsilver-based coatings. The first mirror 1014 a, the second mirror 1014b, and/or the third mirror 1014 c may be controlled with a computersystem 1101. The computer system 1101 may turn the first mirror 1014 a,the second mirror 1014 b, and/or the third mirror 1014 c “on” or “off”in order to re-direct the expanded laser beam 1054 as desired.

The dichroic mirror may be a short pass dichroic mirror. The long passdichroic mirror 1024 may reflect the expanded laser beam 1054 into thefocusing objective 1032. In some instances, a beam combiner may be usedto re-direct the expanded laser beam 1054 into the focusing objective1032 instead of using the long pass dichroic mirror 1024. The long passdichroic mirror 1024 may be controlled with a computer system 1101 tore-direct the expanded laser beam 1054 into the focusing objective 1032.The focusing objective 1032 may concentrate the expanded laser beam 1054as it is projected into the printing chamber 1034. The printing chamber1034 may be a media chamber 122. The printing chamber 1034 may comprisea cell-containing medium, a plurality of cells, cell constituents (e.g.,organelles), and/or at least one polymer precursor.

A light-emitting diode (LED) collimator 1040 may be used as a source ofcollimated LED light 1056. The LED collimator 1040 may comprise acollimating lens and an LED emitter. The LED may be an inorganic LED, ahigh brightness LED, a quantum dot LED, or an organic LED. The LED maybe a single color LED, a bi-color LED, or a tri-color LED. The LED maybe a blue LED, an ultraviolet LED, a white LED, an infrared LED, a redLED, an orange LED, a yellow LED, a green LED, a violet LED, a pink LED,or a purple LED. The LED collimator 1040 may project a beam ofcollimated LED light 1056 through an f4 lens 1038. The f4 lens 1038 maybe a focusing lens. Once the collimated LED light 1056 is transmittedthrough the f4 lens 1038, the collimated LED light 1056 may be directedinto a light focusing objective 1036. The light focusing objective 1036may focus the collimated LED light 1056 into the printing chamber 1034.The light focusing objective 1036 may focus the collimated LED light1056 in the sample medium. The light focusing objective 1036 may focusthe collimated LED light 1056 in the cell-containing medium. Thecollimated LED light 1056 may be transmitted through the printingchamber 1034 and into the focusing objective 1032. Once the collimatedLED light 1056 exits the focusing objective 1032, the collimated LEDlight 1056 may be directed onto the long pass dichroic mirror 1024. Thecollimated LED light 1056 that is reflected off of the long passdichroic mirror 1024 may be the sample emission 1026. The long passdichroic mirror 1024 may re-direct the sample emission 1026 into an f3lens 1028. The f3 lens 1028 may be a focusing lens. Once sample emission1026 is transmitted through the f3 lens 1028, a detection system 1030detects and/or collects the sample emission 1026 for imaging. Thedetection system 1030 may comprise at least one photomultiplier tube(PMT). The detection system 1030 may comprise at least one camera. Thecamera may be a complementary metal-oxide semiconductor (CMOS) camera, ascientific CMOS camera, a charge-coupled device (CCD) camera, or anelectron-multiplying charge-coupled device (EM-CCD). The detectionsystem 1030 may comprise at least one array-based detector.

FIG. 45 illustrates the optical components and the optical path of yetanother embodiment of the three-dimensional printing system. The opticalcomponents and the optical path shown in FIG. 45 provide athree-dimensional printing system that may use temporal focusing. Thethree-dimensional printing system may comprise an energy source 1100.The energy source 1100 may be a coherent light source. The energy source1100 may be a laser light. The energy source 1100 may be a femto-secondpulsed laser light source. The energy source 1100 may be a first lasersource 140 a, a second laser source 140 b, or a third laser source 140c. The energy source 1100 may be a multi-photon laser beam 120. Theenergy source 1100 may be a two-photon laser beam. The energy source1100 may be controlled by a computer system 1101. The energy source 1100may be tuned by a computer system 1101. The computer system 1101 maycontrol and/or set the energy wavelength of the energy source 1100 priorto or during the printing process. They computer system 1101 may producedifferent excitation wavelengths by setting the wavelength of the energysource 1100.

The energy source 1100 may be pulsed. The energy source 1100 may bepulsed at a rate of about 500 kilohertz (kHz). The energy source 1100(e.g., laser) may provide energy (e.g., laser beam) having energypackets with pulsed energies (per packet) from about at least 1 microjoule (μJ) to 1,000,000 μJ. The energy source 1100 (e.g., laser) mayprovide energy (e.g., laser beam) having energy packets with pulsedenergies (per packet) from about at least 1 micro joule (μJ) to 100,000μL or more. The energy source 1100 (e.g., laser) may provide energy(e.g., laser beam) having energy packets with pulsed energies (perpacket) from about at least 1 micro joule (μJ) to 1,000 μL or more. Theenergy source 1100 (e.g., laser) may provide energy (e.g., laser beam)having energy packets with pulsed energies (per packet) from about atleast 1 micro joule (μJ) to 100 μL or more. The energy source 1100(e.g., laser) may provide energy (e.g., laser beam) having energypackets with pulsed energies (per packet) from about at least 10 microjoule (μJ) to 100 μL or more. The energy source 1100 (e.g., laser) mayprovide energy (e.g., laser beam) having energy packets with pulsedenergies (per packet) from about at least 1 micro joule (μJ) to 50 μL ormore. The energy source 1100 (e.g., laser) may provide energy (e.g.,laser beam) having energy packets with pulsed energies (per packet) fromabout at least 1 micro joule (μJ) to 20 μL or more. The energy source1100 (e.g., laser) may provide energy (e.g., laser beam) having energypackets with pulsed energies (per packet) from about at least 1 microjoule (μJ) to 50 μL or more. The energy source 1100 (e.g., laser) mayprovide energy (e.g., laser beam) having energy packets with pulsedenergies (per packet) from about at least 40 micro joule (μJ) to 80 μLor more. The energy source 1100 (e.g., laser) may provide energy (e.g.,laser beam) having energy packets with pulsed energies (per packet) fromabout at least 120 micro joule (μJ) to 160 μL or more.

The energy source 1100 (e.g., laser) may provide energy (e.g., laserbeam) having energy packets with pulsed energies (per packet) of about10 μJ. The energy source 1100 (e.g., laser) may provide energy (e.g.,laser beam) having energy packets with pulsed energies (per packet) ofabout 20 μJ. The energy source 1100 (e.g., laser) may provide energy(e.g., laser beam) having energy packets with pulsed energies (perpacket) of about 30 μJ. The energy source 1100 (e.g., laser) may provideenergy (e.g., laser beam) having energy packets with pulsed energies(per packet) of about 40 μJ. The energy source 1100 (e.g., laser) mayprovide energy (e.g., laser beam) having energy packets with pulsedenergies (per packet) of about 50 μJ. The energy source 1100 (e.g.,laser) may provide energy (e.g., laser beam) having energy packets withpulsed energies (per packet) of about 60 μJ. The energy source 1100(e.g., laser) may provide energy (e.g., laser beam) having energypackets with pulsed energies (per packet) of about 70 μJ. The energysource 1100 (e.g., laser) may provide energy (e.g., laser beam) havingenergy packets with pulsed energies (per packet) of about 80 μJ. Theenergy source 1100 (e.g., laser) may provide energy (e.g., laser beam)having energy packets with pulsed energies (per packet) of about 90 μJ.The energy source 1100 (e.g., laser) may provide energy (e.g., laserbeam) having energy packets with pulsed energies (per packet) of about100 μJ. The energy source 1100 (e.g., laser) may provide energy (e.g.,laser beam) having energy packets with pulsed energies (per packet) ofabout 110 μJ. The energy source 1100 (e.g., laser) may provide energy(e.g., laser beam) having energy packets with pulsed energies (perpacket) of about 120 μJ. The energy source 1100 (e.g., laser) mayprovide energy (e.g., laser beam) having energy packets with pulsedenergies (per packet) of about 130 μJ. The energy source 1100 (e.g.,laser) may provide energy (e.g., laser beam) having energy packets withpulsed energies (per packet) of about 140 μJ. The energy source 1100(e.g., laser) may provide energy (e.g., laser beam) having energypackets with pulsed energies (per packet) of about 150 μJ. The energysource 1100 (e.g., laser) may provide energy (e.g., laser beam) havingenergy packets with pulsed energies (per packet) of about 160 μJ. Theenergy source 1100 (e.g., laser) may provide energy (e.g., laser beam)having energy packets with pulsed energies (per packet) of about 170 μJ.The energy source 1100 (e.g., laser) may provide energy (e.g., laserbeam) having energy packets with pulsed energies (per packet) of about180 μJ. The energy source 1100 (e.g., laser) may provide energy (e.g.,laser beam) having energy packets with pulsed energies (per packet) ofabout 190 μJ. The energy source 1100 (e.g., laser) may provide energy(e.g., laser beam) having energy packets with pulsed energies (perpacket) of about 200 μJ. The energy source 1100 (e.g., laser) mayprovide energy (e.g., laser beam) having energy packets with pulsedenergies (per packet) of about 20,000 μJ. The energy source 1100 (e.g.,laser) may provide energy (e.g., laser beam) having energy packets withpulsed energies (per packet) of about 100,000 μJ. The energy source 1100(e.g., laser) may provide energy (e.g., laser beam) having energypackets with pulsed energies (per packet).

The energy source 1100 (e.g., laser) may provide energy (e.g., laserbeam) having a wavelength from about 300 nm to 5 mm, 600 nm to 1500 nm,350 nm to 1800 nm, or 1800 nm to 5 mm. The energy source 1100 (e.g.,laser) may provide energy (e.g., laser beam) having a wavelength of atleast about 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1mm, 1.1 mm, 1.2, mm, 1.3 mm, 1.4 mm, 1.5 mm, 1.6 mm, 1.7 mm, 1.8 mm, 1.9mm, 2 mm, 3 mm, 4 mm, 5 mm, or greater.

As shown in FIG. 45, the energy source 1100 may project a laser beam1102 through a shutter 1104. Once the laser beam 1102 exits the shutter1104, the laser beam 1102 may be directed through a rotating half-waveplate 1106. The rotating half-wave plate 1106 may alter the polarizationstate of the laser beam 1102 such that the difference in phase delaybetween the two linear polarization directions is π. The difference inphase delay may correspond to a propagation phase shift over a distanceof λ/2. Other types of wave plates may be utilized with the systemdisclosed herein; for example, a rotating quarter-wave plate may beused. The rotating half-wave plate 1106 may be a true zero-order waveplate, a low order wave plate, or a multiple-order wave plate. Therotating half-wave plate 1106 may be composed of crystalline quartz(SiO₂), calcite (CaCO₃), magnesium fluoride (MgF₂), sapphire (Al₂O₃),mica, or a birefringent polymer.

The laser beam 1102 may exit the rotating half-wave plate 1106 and maybe directed through a polarizing beam splitter 1108. The polarizing beamsplitter 1108 may split the laser beam 1102 into a first laser beam 1102a and a second laser beam 1102 b. The first laser beam 1102 a may bedirected to a beam dump 1110. The beam dump 1110 is an optical elementthat may be used to absorb stray portions of a laser beam. The beam dump1110 may absorb the first laser beam 1102 a. The first laser beam 1102 amay be a stray laser beam. The beam dump 1110 may absorb the secondlaser beam 1102 b. The second laser beam 1102 b may be a stray laserbeam. The laser beam 1102 may be directed into the beam dump 1110 in itsentirety and thus, may serve as a default “off” state of the printingsystem. The second laser beam 1102 b may be directed to a beam expander1112. The beam expander 1112 may expand the size of the second laserbeam 1102 b. The beam expander 1112 may increase the diameter of theinput, second laser beam 1102 b to a larger diameter of an output,expanded laser beam 1154. The beam expander 1112 may be a prismatic beamexpander. The beam expander 1112 may be a telescopic beam expander. Thebeam expander 1112 may be a multi-prism beam expander. The beam expander1112 may be a Galilean beam expander. The beam expander 1112 may providea beam expander power of about 2×, 3×, 5×, 10×, 20×, or 40×. The beamexpander 1112 may provide a beam expander power ranging from about 2× toabout 5×. The beam expander 1112 may provide continuous beam expansionbetween about 2× and about 5×. The beam expander 1112 may provide a beamexpander power ranging from about 5× to about 10×. The beam expander1112 may provide continuous beam expansion between about 5× and about10×. The expanded laser beam 1154 may be collimated upon exiting thebeam expander 1112.

After exiting the beam expander 1112, the expanded laser beam 1154 maybe directed to a first mirror 1114 a, which may re-direct the expandedlaser beam 1154 to a first spatial light modulator (SLM) 1116 a. Afterexiting the first SLM 1116, the expanded laser beam 1154 may be directedto an f1 lens 1118. The f1 lens 1118 may be a focusing lens. Afterexiting the f1 lens, the expanded laser beam 1154 may be directed to agrating 1142. The grating 1142 may be a diffractive laser beam splitter.The grating 1142 may be a holographic grating. The grating 1142 may be aruled grating. The grating 1142 may be a subwavelength grating. Thegrating 1142 may split and/or diffract the expanded laser beam 1154 intoa plurality of expanded laser beams (not shown in FIG. 45). The grating1142 may act as a dispersive element. Once the expanded laser beam 1154is split, diffracted, and/or dispersed by the grating 1142, the expandedlaser beam 1154 may be transmitted through an f2 lens 1122. The f2 lens1122 may be a focusing lens. After exiting the f2 lens 1122, theexpanded laser beam 1154 may be directed to a second SLM 1116 b. TheSLMs (i.e., the first SLM 1116 a and the second SLM 1116 b) may becontrolled by a computer system 1101. The SLMs may perform all of thefunctions, as described supra, of the SLM 1016 presented in FIG. 44.

After exiting the second SLM 1116 b, the expanded laser beam 1154 may bedirected to an f3 lens 1128. The f3 lens 1128 may be a focusing lens.After exiting the f3 lens, the expanded laser beam 1154 may be directedto blocking element 1120. The blocking element 1120 may be immovable.The blocking element 1120 may be used to suppress illumination from azero-order spot. After exiting the blocking element 1120, the expandedenergy beam 1154 may be directed through an f4 lens 1138. The f4 lens1138 may be a focusing lens. After exiting the f4 lens 1138, theexpanded laser beam 1154 may be directed onto a second mirror 1114 b andmay be subsequently directed onto a third mirror 1114 c. The thirdmirror 1114 c may re-direct the expanded laser beam 1154 through a longpass dichroic mirror 1124. The first mirror 1114 a, the second mirror1114 b, and/or the third mirror 1114 c may be controlled with a computersystem 1101. The computer system 1101 may turn the first mirror 1114 a,the second mirror 1114 b, and/or the third mirror 1114 c “on” or “off”in order to re-direct the expanded laser beam 1154 as desired. Thedichroic mirror may be a short pass dichroic mirror. The long passdichroic mirror 1124 may reflect the expanded laser beam 1154 into thefocusing objective 1132. In some instances, a beam combiner may be usedto re-direct the expanded laser beam 1154 into the focusing objective1132 instead of using the long pass dichroic mirror 1124. The long passdichroic mirror 1124 may be controlled with a computer system 1101 tore-direct the expanded laser beam 1154 into the focusing objective 1132.The focusing objective 1132 may concentrate the expanded laser beam 1154as it is projected into the printing chamber 1134. The printing chamber1134 may be a media chamber 122. The printing chamber 1134 may comprisea cell-containing medium, a plurality of cells, cell constituents (e.g.,organelles), and/or at least one polymer precursor.

The printing chamber 1134 may be mounted on a movable stage 1146. Themovable stage 1146 may be an xy stage, a z stage, and/or an xyz stage.The movable stage 1146 may be manually positioned. The movable stage1146 may be automatically positioned. The movable stage 1146 may be amotorized stage. The movable stage 1146 may be controlled by thecomputer system 1101. The computer system 1101 may control the movementof the movable stage 1146 in the x, y, and/or z directions. The computersystem 1101 may automatically position the movable stage 1146 in adesired x, y, and/or z position. The computer system 1101 may positionthe movable stage 1146 in a desired x, y, and/or z position with apositional accuracy of at most about 3 μm. The computer system 1101 mayposition the movable stage 1146 in a desired x, y, and/or z positionwith a positional accuracy of at most about 2 μm. The computer system1101 may position the movable stage 1146 in a desired x, y, and/or zposition with a positional accuracy of at most about 1 μm. The computersystem 1101 may automatically adjust the position of the movable stage1146 prior or during three-dimensional printing. The computer system1101 may comprise a piezoelectric (piezo) controller to providecomputer-controlled z-axis (i.e., vertical direction) positioning andactive location feedback. The computer system 1101 may comprise ajoystick console to enable a user to control a position of the movablestage 1146. The joystick console may be a z-axis console and/or anx-axis and y-axis console. The movable stage 1146 may comprise aprinting chamber holder. The printing chamber holder may be a bracket, aclip, and/or a recessed sample holder. The movable stage 1146 maycomprise a multi-slide holder, a slide holder, and/or a petri dishholder. The movable stage 1146 may comprise a sensor to provide locationfeedback. The sensor may be a capacitive sensor. The sensor may be apiezoresistive sensor. The movable stage 1146 may comprise at least oneactuator (e.g., piezoelectric actuator) that moves (or positions) themovable stage 1146.

A light-emitting diode (LED) collimator 1140 may be used as a source ofcollimated LED light 1156. The LED collimator 1140 may comprise acollimating lens and an LED emitter. The LED may be an inorganic LED, ahigh brightness LED, a quantum dot LED, or an organic LED. The LED maybe a single color LED, a bi-color LED, or a tri-color LED. The LED maybe a blue LED, an ultraviolet LED, a white LED, an infrared LED, a redLED, an orange LED, a yellow LED, a green LED, a violet LED, a pink LED,or a purple LED. The LED collimator 1140 may project a beam ofcollimated LED light 1156 through an f6 lens 1148. The f6 lens 1148 maybe a focusing lens. Once the collimated LED light 1156 is transmittedthrough the f6 lens 1148, the collimated LED light 1156 may be directedinto a light focusing objective 1136. The light focusing objective 1136may focus the collimated LED light 1156 into the printing chamber 1134.The light focusing objective 1136 may focus the collimated LED light1156 in the sample medium. The light focusing objective 1136 may focusthe collimated LED light 1156 in the cell-containing medium. Thecollimated LED light 1156 may be transmitted through the printingchamber 1134 and into the focusing objective 1132. Once the collimatedLED light 1156 exits the focusing objective 1132, the collimated LEDlight 1156 may be directed onto the long pass dichroic mirror 1124. Thecollimated LED light 1156 that is reflected off of the long passdichroic mirror 1124 may be the sample emission 1126. The long passdichroic mirror 1124 may re-direct the sample emission 1126 into an f5lens 1144. The f5 lens 1144 may be a focusing lens. Once sample emission1126 is transmitted through the f5 lens 1144, a detection system 1130detects and/or collects the sample emission 1126 for imaging. Thedetection system 1130 may comprise at least one photomultiplier tube(PMT). The detection system 1130 may comprise at least one camera. Thecamera may be a complementary metal-oxide semiconductor (CMOS) camera, ascientific CMOS camera, a charge-coupled device (CCD) camera, or anelectron-multiplying charge-coupled device (EM-CCD). The detectionsystem 1130 may comprise at least one array-based detector.

FIG. 46 illustrates the optical components and the optical path of anadditional embodiment of the three-dimensional printing system. Theoptical components and the optical path shown in FIG. 46 provide athree-dimensional printing system that may not use temporal focusing.The three-dimensional printing system may comprise an energy source1200. The energy source 1200 may be a coherent light source. The energysource 1200 may be a laser light. The energy source 1200 may be afemto-second pulsed laser light source. The energy source 1200 may be afirst laser source 140 a, a second laser source 140 b, or a third lasersource 140 c. The energy source 1200 may be a multi-photon laser beam120. The energy source 1200 may be controlled by a computer system 1101.The energy source 1200 may be tuned by a computer system 1101. Thecomputer system 1101 may control and/or set the energy wavelength of theenergy source 1200 prior to or during the printing process. Theycomputer system 1101 may produce different excitation wavelengths bysetting the wavelength of the energy source 1200.

The energy source 1200 may be pulsed. The energy source 1200 may bepulsed at a rate of about 500 kilohertz (kHz). The energy source 1200(e.g., laser) may provide energy (e.g., laser beam) having energypackets with pulsed energies (per packet) from about at least 1 microjoule (μJ) to 1,000,000 μJ. The energy source 1200 (e.g., laser) mayprovide energy (e.g., laser beam) having energy packets with pulsedenergies (per packet) from about at least 1 micro joule (μJ) to 100,000μL or more. The energy source 1200 (e.g., laser) may provide energy(e.g., laser beam) having energy packets with pulsed energies (perpacket) from about at least 1 micro joule (μJ) to 1,000 μL or more. Theenergy source 1200 (e.g., laser) may provide energy (e.g., laser beam)having energy packets with pulsed energies (per packet) from about atleast 1 micro joule (μJ) to 100 μL or more. The energy source 1200(e.g., laser) may provide energy (e.g., laser beam) having energypackets with pulsed energies (per packet) from about at least 10 microjoule (μJ) to 100 μL or more. The energy source 1200 (e.g., laser) mayprovide energy (e.g., laser beam) having energy packets with pulsedenergies (per packet) from about at least 1 micro joule (μJ) to 50 μL ormore. The energy source 1200 (e.g., laser) may provide energy (e.g.,laser beam) having energy packets with pulsed energies (per packet) fromabout at least 1 micro joule (μJ) to 20 μL or more. The energy source1200 (e.g., laser) may provide energy (e.g., laser beam) having energypackets with pulsed energies (per packet) from about at least 1 microjoule (μJ) to 50 μL or more. The energy source 1200 (e.g., laser) mayprovide energy (e.g., laser beam) having energy packets with pulsedenergies (per packet) from about at least 40 micro joule (μJ) to 80 μLor more. The energy source 1200 (e.g., laser) may provide energy (e.g.,laser beam) having energy packets with pulsed energies (per packet) fromabout at least 120 micro joule (μJ) to 160 μL or more.

The energy source 1200 (e.g., laser) may provide energy (e.g., laserbeam) having energy packets with pulsed energies (per packet) of about10 μJ. The energy source 1200 (e.g., laser) may provide energy (e.g.,laser beam) having energy packets with pulsed energies (per packet) ofabout 20 μJ. The energy source 1200 (e.g., laser) may provide energy(e.g., laser beam) having energy packets with pulsed energies (perpacket) of about 30 μJ. The energy source 1200 (e.g., laser) may provideenergy (e.g., laser beam) having energy packets with pulsed energies(per packet) of about 40 μJ. The energy source 1200 (e.g., laser) mayprovide energy (e.g., laser beam) having energy packets with pulsedenergies (per packet) of about 50 μJ. The energy source 1200 (e.g.,laser) may provide energy (e.g., laser beam) having energy packets withpulsed energies (per packet) of about 60 μJ. The energy source 1200(e.g., laser) may provide energy (e.g., laser beam) having energypackets with pulsed energies (per packet) of about 70 μJ. The energysource 1200 (e.g., laser) may provide energy (e.g., laser beam) havingenergy packets with pulsed energies (per packet) of about 80 μJ. Theenergy source 1200 (e.g., laser) may provide energy (e.g., laser beam)having energy packets with pulsed energies (per packet) of about 90 μJ.The energy source 1200 (e.g., laser) may provide energy (e.g., laserbeam) having energy packets with pulsed energies (per packet) of about100 μJ. The energy source 1200 (e.g., laser) may provide energy (e.g.,laser beam) having energy packets with pulsed energies (per packet) ofabout 110 μJ. The energy source 1200 (e.g., laser) may provide energy(e.g., laser beam) having energy packets with pulsed energies (perpacket) of about 120 μJ. The energy source 1200 (e.g., laser) mayprovide energy (e.g., laser beam) having energy packets with pulsedenergies (per packet) of about 130 μJ. The energy source 1200 (e.g.,laser) may provide energy (e.g., laser beam) having energy packets withpulsed energies (per packet) of about 140 μJ. The energy source 1200(e.g., laser) may provide energy (e.g., laser beam) having energypackets with pulsed energies (per packet) of about 150 μJ. The energysource 1200 (e.g., laser) may provide energy (e.g., laser beam) havingenergy packets with pulsed energies (per packet) of about 160 μJ. Theenergy source 1200 (e.g., laser) may provide energy (e.g., laser beam)having energy packets with pulsed energies (per packet) of about 170 μJ.The energy source 1200 (e.g., laser) may provide energy (e.g., laserbeam) having energy packets with pulsed energies (per packet) of about180 μJ. The energy source 1200 (e.g., laser) may provide energy (e.g.,laser beam) having energy packets with pulsed energies (per packet) ofabout 190 μJ. The energy source 1200 (e.g., laser) may provide energy(e.g., laser beam) having energy packets with pulsed energies (perpacket) of about 200 μJ. The energy source 1200 (e.g., laser) mayprovide energy (e.g., laser beam) having energy packets with pulsedenergies (per packet) of about 20,000 μJ. The energy source 1200 (e.g.,laser) may provide energy (e.g., laser beam) having energy packets withpulsed energies (per packet) of about 100,000 μJ. The energy source 1200(e.g., laser) may provide energy (e.g., laser beam) having energypackets with pulsed energies (per packet).

The energy source 1200 (e.g., laser) may provide energy (e.g., laserbeam) having a wavelength from e.g. about at least 300 nm to about 5 mmor more. The energy source 1200 (e.g., laser) may provide energy (e.g.,laser beam) having a wavelength of about at least 600 to about 1500 nmor more. The energy source 1200 (e.g., laser) may provide energy (e.g.,laser beam) having a wavelength from about at least 350 nm to about 1800nm or more. The energy source 1200 (e.g., laser) may provide energy(e.g., laser beam) having a wavelength from about at least 1800 nm toabout 5 mm or more. The energy source 1200 (e.g., laser) may provideenergy (e.g., laser beam) having a wavelength of about 300 nm. Theenergy source 1200 (e.g., laser) may provide energy (e.g., laser beam)having a wavelength of about 400 nm. The energy source 1200 (e.g.,laser) may provide energy (e.g., laser beam) having a wavelength ofabout 600 nm. The energy source 1200 (e.g., laser) may provide energy(e.g., laser beam) having a wavelength of about 700 nm. The energysource 1200 (e.g., laser) may provide energy (e.g., laser beam) having awavelength of about 800 nm. The energy source 1200 (e.g., laser) mayprovide energy (e.g., laser beam) having a wavelength of about 900 nm.The energy source 1200 (e.g., laser) may provide energy (e.g., laserbeam) having a wavelength of about 1200 nm. The energy source 1200(e.g., laser) may provide energy (e.g., laser beam) having a wavelengthof about 1200 nm. The energy source 1200 (e.g., laser) may provideenergy (e.g., laser beam) having a wavelength of about 1200 nm. Theenergy source 1200 (e.g., laser) may provide energy (e.g., laser beam)having a wavelength of about 1300 nm. The energy source 1200 (e.g.,laser) may provide energy (e.g., laser beam) having a wavelength ofabout 1400 nm. The energy source 1200 (e.g., laser) may provide energy(e.g., laser beam) having a wavelength of about 1500 nm. The energysource 1200 (e.g., laser) may provide energy (e.g., laser beam) having awavelength of about 1600 nm. The energy source 1200 (e.g., laser) mayprovide energy (e.g., laser beam) having a wavelength of about 1700 nm.The energy source 1200 (e.g., laser) may provide energy (e.g., laserbeam) having a wavelength of about 1800 nm. The energy source 1200(e.g., laser) may provide energy (e.g., laser beam) having a wavelengthof about 1900 nm. The energy source 1200 (e.g., laser) may provideenergy (e.g., laser beam) having a wavelength of about 2000 nm. Theenergy source 1200 (e.g., laser) may provide energy (e.g., laser beam)having a wavelength of about 3000 nm. The energy source 1200 (e.g.,laser) may provide energy (e.g., laser beam) having a wavelength ofabout 4000 nm. The energy source 1200 (e.g., laser) may provide energy(e.g., laser beam) having a wavelength of about 5000 nm.

As shown in FIG. 46, the energy source 1200 may project a laser beam1202 through a shutter 1104. Once the laser beam 1202 exits the shutter1204, the laser beam 1202 may be directed through a rotating half-waveplate 1206. The rotating half-wave plate 1206 may alter the polarizationstate of the laser beam 1202 such that the difference in phase delaybetween the two linear polarization directions is π. The difference inphase delay may correspond to a propagation phase shift over a distanceof λ/2. Other types of wave plates may be utilized with the systemdisclosed herein; for example, a rotating quarter-wave plate may beused. The rotating half-wave plate 1206 may be a true zero-order waveplate, a low order wave plate, or a multiple-order wave plate. Therotating half-wave plate 1206 may be composed of crystalline quartz(SiO₂), calcite (CaCO₃), magnesium fluoride (MgF₂), sapphire (Al₂O₃),mica, or a birefringent polymer.

The laser beam 1202 may exit the rotating half-wave plate 1206 and maybe directed through a polarizing beam splitter 1208. The polarizing beamsplitter 1208 may split the laser beam 1202 into a first laser beam 1202a and a second laser beam 1202 b. The first laser beam 1202 a may bedirected to a beam dump 1210. The beam dump 1210 is an optical elementthat may be used to absorb stray portions of a laser beam. The beam dump1210 may absorb the first laser beam 1202 a. The first laser beam 1202 amay be a stray laser beam. The beam dump 1210 may absorb the secondlaser beam 1202 b. The second laser beam 1202 b may be a stray laserbeam. The laser beam 1202 may be directed into the beam dump 1210 in itsentirety and thus, may serve as a default “off” state of the printingsystem. The second laser beam 1202 b may be directed to a beam expander1212. The beam expander 1212 may expand the size of the second laserbeam 1202 b. The beam expander 1212 may increase the diameter of theinput, second laser beam 1202 b to a larger diameter of an output,expanded laser beam 1254. The beam expander 1212 may be a prismatic beamexpander. The beam expander 1212 may be a telescopic beam expander. Thebeam expander 1212 may be a multi-prism beam expander. The beam expander1212 may be a Galilean beam expander. The beam expander 1212 may providea beam expander power of about 2×, 3×, 5×, 10×, 20×, or 40×. The beamexpander 1212 may provide a beam expander power ranging from about 2× toabout 5×. The beam expander 1212 may provide continuous beam expansionbetween about 2× and about 5×. The beam expander 1212 may provide a beamexpander power ranging from about 5× to about 10×. The beam expander1212 may provide continuous beam expansion between about 5× and about10×. The expanded laser beam 1254 may be collimated upon exiting thebeam expander 1212.

After exiting the beam expander 1212, the expanded laser beam 1254 maybe directed to a first mirror 1214 a, which may re-direct the expandedlaser beam 1254 to a first spatial light modulator (SLM) 1216 a. Afterexiting the first SLM 1216, the expanded laser beam 1254 may be directedto an f1 lens 1218. The f1 lens 1218 may be a focusing lens. Afterexiting the f1 lens, the expanded laser beam 1254 may be directed to amirror with blocking element 1250. The mirror with blocking element 1250may be used to suppress illumination from a zero-order spot.

Once the expanded laser beam 1254 is reflected by the mirror withblocking element 1250, the expanded laser beam 1254 may be transmittedthrough an f2 lens 1222. The f2 lens 1222 may be a focusing lens. Afterexiting the f2 lens 1222, the expanded laser beam 1254 may be directedto a second SLM 1216 b. The SLMs (i.e., the first SLM 1216 a and thesecond SLM 1216 b) may be controlled by a computer system 1101. The SLMsmay perform all of the functions, as described supra, of the SLM 1016and the SLM 1116, as presented in FIGS. 44 and 45, respectively.

After exiting the second SLM 1216 b, the expanded laser beam 1254 may bedirected to an f3 lens 1228. After exiting the f3 lens, the expandedlaser beam 1254 may be directed to blocking element 1220. The blockingelement 1220 may be immovable. The blocking element 1220 may be used tosuppress illumination from a zero-order spot. After exiting the blockingelement 1220, the expanded energy beam 1254 may be directed through anf4 lens 1238. The f4 lens 1238 may be a focusing lens. After exiting thef4 lens 1238, the expanded laser beam 1254 may be directed onto a secondmirror 1214 b and may be subsequently directed onto a third mirror 1214c. The third mirror 1214 c may re-direct the expanded laser beam 1254through a long pass dichroic mirror 1224. The first mirror 1214 a, thesecond mirror 1214 b, and/or the third mirror 1214 c may be controlledwith a computer system 1101. The computer system 1101 may turn the firstmirror 1214 a, the second mirror 1214 b, and/or the third mirror 1214 c“on” or “off” in order to re-direct the expanded laser beam 1254 asdesired. The dichroic mirror may be a short pass dichroic mirror. Thelong pass dichroic mirror 1224 may reflect the expanded laser beam 1254into the focusing objective 1232. In some instances, a beam combiner maybe used to re-direct the expanded laser beam 1254 into the focusingobjective 1232 instead of using the long pass dichroic mirror 1224. Thelong pass dichroic mirror 1224 may be controlled with a computer system1101 to re-direct the expanded laser beam 1254 into the focusingobjective 1232. The focusing objective 1232 may concentrate the expandedlaser beam 1254 as it is projected into the printing chamber 1234. Theprinting chamber 1234 may be a media chamber 122. The printing chamber1234 may comprise a cell-containing medium, a plurality of cells, cellconstituents (e.g., organelles), and/or at least one polymer precursor.

The printing chamber 1234 may be mounted on a movable stage 1246. Themovable stage 1246 may be an xy stage, a z stage, and/or an xyz stage.The movable stage 1246 may be manually positioned. The movable stage1246 may be automatically positioned. The movable stage 1246 may be amotorized stage. The movable stage 1246 may be controlled by thecomputer system 1101. The computer system 1101 may control the movementof the movable stage 1246 in the x, y, and/or z directions. The computersystem 1101 may automatically position the movable stage 1246 in adesired x, y, and/or z position. The computer system 1101 may positionthe movable stage 1246 in a desired x, y, and/or z position with apositional accuracy of at most about 3 μm. The computer system 1101 mayposition the movable stage 1246 in a desired x, y, and/or z positionwith a positional accuracy of at most about 2 μm. The computer system1101 may position the movable stage 1246 in a desired x, y, and/or zposition with a positional accuracy of at most about 1 μm. The computersystem 1101 may automatically adjust the position of the movable stage1246 prior or during three-dimensional printing. The computer system1101 may comprise a piezo controller to provide computer-controlledz-axis (i.e., vertical direction) positioning and active locationfeedback. The computer system 1101 may comprise a joystick console toenable a user to control a position of the movable stage 1246. Thejoystick console may be a z-axis console and/or an x-axis and y-axisconsole. The movable stage 1246 may comprise a printing chamber holder.The printing chamber holder may be a bracket, a clip, and/or a recessedsample holder. The movable stage 1246 may comprise a multi-slide holder,a slide holder, and/or a petri dish holder. The movable stage 1246 maycomprise a sensor to provide location feedback. The sensor may be acapacitive sensor. The sensor may be a piezoresistive sensor. Themovable stage 1246 may comprise at least one actuator (e.g.,piezoelectric actuator) that moves (or positions) the movable stage1246.

A light-emitting diode (LED) collimator 1240 may be used as a source ofcollimated LED light 1256. The LED collimator 1240 may comprise acollimating lens and an LED emitter. The LED may be an inorganic LED, ahigh brightness LED, a quantum dot LED, or an organic LED. The LED maybe a single color LED, a bi-color LED, or a tri-color LED. The LED maybe a blue LED, an ultraviolet LED, a white LED, an infrared LED, a redLED, an orange LED, a yellow LED, a green LED, a violet LED, a pink LED,or a purple LED. The LED collimator 1240 may project a beam ofcollimated LED light 1256 through an f6 lens 1248. The f6 lens 1248 maybe a focusing lens. Once the collimated LED light 1256 is transmittedthrough the f6 lens 1248, the collimated LED light 1156 may be directedinto a light focusing objective 1236. The light focusing objective 1236may focus the collimated LED light 1256 into the printing chamber 1234.The light focusing objective 1236 may focus the collimated LED light1256 in the sample medium. The light focusing objective 1236 may focusthe collimated LED light 1256 in the cell-containing medium. Thecollimated LED light 1256 may be transmitted through the printingchamber 1234 and into the focusing objective 1232. Once the collimatedLED light 1256 exits the focusing objective 1232, the collimated LEDlight 1256 may be directed onto the long pass dichroic mirror 1224. Thecollimated LED light 1256 that is reflected off of the long passdichroic mirror 1224 may be the sample emission 1226. The long passdichroic mirror 1224 may re-direct the sample emission 1226 into an f5lens 1244. The f5 lens may be a focusing lens. Once sample emission 1226is transmitted through the f5 lens 1244, a detection system 1230 detectsand/or collects the sample emission 1226 for imaging. The detectionsystem 1230 may comprise at least one photomultiplier tube (PMT). Thedetection system 1230 may comprise at least one camera. The camera maybe a complementary metal-oxide semiconductor (CMOS) camera, a scientificCMOS camera, a charge-coupled device (CCD) camera, or anelectron-multiplying charge-coupled device (EM-CCD). The detectionsystem 1230 may comprise at least one array-based detector.

FIG. 47 illustrates a light detection system 1330. The light detectionsystem 1330 may comprise a plurality of long pass dichroic mirrorsarranged in series. The light detection system 1330 may comprise aplurality of long pass dichroic mirrors arranged in parallel. The lightdetection system 1330 may comprise a plurality of long pass dichroicmirrors arranged in series and parallel. As shown in FIGS. 44-46, theoptical paths may comprise an LED collimator that projects a beam ofcollimated LED light 1356 onto the focusing objectives. Once thecollimated LED light 1356 is reflected from the first long pass dichroicmirror 1324 a, the collimated LED light 1356 may be converted to asample emission 1326. The sample emission 1326 may be directed throughan f5 lens 1344. The f5 lens 1344 may be a focusing lens. After thesample emission 1326 exits the f5 lens 1344, the sample emission 1326may be directed to a series of long pass dichroic mirrors comprising asecond long pass dichroic mirror 1324 b, a third long pass dichroicmirror 1324 c, a fourth long pass dichroic mirror 1324 d, and a fifthlong pass dichroic mirror 1324 e, as shown in FIG. 47. The sampleemission 1326 may be reflected off of the second long pass dichroicmirror 1324 b and onto a first light detector 1352 a. The sampleemission 1326 may be reflected off of the third long pass dichroicmirror 1324 c and onto a second light detector 1352 b. The sampleemission 1326 may be reflected off of the fourth long pass dichroicmirror 1324 d and onto a third light detector 1352 c. The sampleemission 1326 may be reflected off of the fifth long pass dichroicmirror 1324 e and onto a fourth light detector 1352 d. The sampleemission 1326 may be reflected off of the fifth long pass dichroicmirror 1324 e and onto a fifth light detector 1352 e. The light detectormay be a photomultiplier tube (PMT). The light detector may be a camera.The light detector may be a complementary metal-oxide semiconductor(CMOS) camera, a scientific CMOS camera, a charge-coupled device (CCD)camera, or an electron-multiplying charge-coupled device (EM-CCD). Thelight detector may be an array-based detector. The light detectionsystem 1330 may comprise a plurality of long pass dichroic mirrors thathave progressively red-shifted cutoff wavelengths. In some instances,the second long pass dichroic mirror 1324 b may have a cutoff wavelengthof about 460 nm, the third long pass dichroic mirror 1324 c may have acutoff wavelength of about 500 nm, the fourth long pass dichroic mirror1324 d may have a cutoff wavelength of about 540 nm, the fifth long passdichroic mirror 1324 e may have a cutoff wavelength of about 570 nm.

The light detection system 1330 may be controlled by the computer system1101. The computer system 1101 may collect and/or process the signalsobtained by the first light detector 1352 a, the second light detector1352 b, the third light detector 1352 c, and the fourth light detector1352 d. The computer system 1101 may provide control feedback to thethree-dimensional printing system based on the light detector signals,of the light detection system 1330, which may be collected and/orprocessed by the computer system 1101. The computer system 1101 may havecontrol feedback over any optical component and/or hardware of theoptical paths described in FIGS. 44-46. The computer system 1101 mayhave control feedback over any optical component and/or hardware of thelight detection system 1330 shown in FIG. 47. The computer system 1101may control, for example, an SLM, a shutter, a movable stage, a mirror,a lens, a focusing objective, a beam expander, an LED collimator, agrating, and/or a blocking element in response to a signal from thelight detection system 1330.

FIG. 5A illustrates an embodiment of the multi-photon tissue print head118. The multi-photon print-head 118 may receive the multi-photon laserbeam 120 (comprising one or more wavelengths) from the laser system 116and may focus the beam 120 through the final optical path with iscomprised of finishing optics that are comprised of an optional scanhead, long pass mirror for use collection and recording of back-scatterlight and a focusing objective 200, projecting the beam 120 into themedia chamber 122. The light may be collected by the same objective asused to print, and then shunted via a long-pass mirror to the single orbank of PMTs, or a CCD camera.

In some designs, the optics may send the laser through a fiber opticcable for easier control of where the light is deposited in the tissueprinting vessel.

The systems disclosed herein can utilize a range of focusing objectives,for example, with an increasingly lower magnification; the field of viewmay be increasingly larger. In some cases, the field of view may be theprint area that the microscope is capable of, in a single projectionarea. In some cases, 5×, 10×, or 20× objectives may be employed. In somecases, objectives with high numerical apertures ranging between at leastabout 0.6 and about 1.2 or more may be employed. The systems disclosedherein may use an objective lens with a magnification ranging from e.g.,about 1× to about 100×. The systems disclosed herein may use anobjective lens with a magnification of about 1×. The systems disclosedherein may use an objective lens with a magnification of about 2×. Thesystems disclosed herein may use an objective lens with a magnificationof about 3×. The systems disclosed herein may use an objective lens witha magnification of about 4×. The systems disclosed herein may use anobjective lens with a magnification of about 10×. The systems disclosedherein may use an objective lens with a magnification of about 20×. Thesystems disclosed herein may use an objective lens with a magnificationof about 40×. The systems disclosed herein may use an objective lenswith a magnification of about 60×. The systems disclosed herein may usean objective lens with a magnification of about 100×.

To maintain structural fidelity of the printed tissues, awater-immersion objective lens may be ideal so as to substantially matchthe angle of incidence within the cell-containing liquid biogel media126. A water-immersion objective lens corrected for refractive indexchanges may be used as printing takes place in liquid media which has asignificantly different refractive index from air.

FIG. 5B illustrates a print head 118 comprising a first objective lens200 a and a second objective lens 200 b. FIG. 5B illustrates invertedoptics for imaging structures. In this embodiment, light may becollected by inverted optics and channeled to a CCD camera, a singlePMT, as shown in FIG. 5B, or a bank of PMTs to create a multi-colorimage. In some embodiments, a second objective head may be inverted andimages may be collected from the underside of the tissue and incidentlight read by PMTs with a series of long pass or band-pass mirrors.

In order for a multi-photon based printer to switch from a printing modeto an imaging mode, x, y raster scanning may be engaged and the DMD orSLM paths may be bypassed or the devices rendered in an off or inactiveposition, or removing them from the light path such that there is only asingle laser line hitting the x, y scanning optics. DMD or SLM paths mayalso in some instances be used for imaging.

Switching to imaging mode may have several uses during the printingprocess: 1) imaging can be used to monitor collagen generation rates ascollagen naturally produces an emission via second harmonic generation,which is a process when two-photon excitation is scanned across thestructures, 2) the edges of printed tissues can be found using imagingmode facilitating the proper linking of blood vessels and other tissuestructures along edges of projection spaces, 3) printed tissuestructures can be validated for structural integrity and fidelity to theprojected images in real-time, and 4) if cells that are temporarilylabeled are used, they can be located within the printed tissues forprocess validation or monitoring.

It may be appreciated that the laser system 116 of the above embodimentsmay have a variety of points of software control including, but notlimited to: The CAD images may be projected by programming changes thatare hardwired to the SLM and/or DMD devices; If TAG lenses are used tocreate a Bessel beam, the current generated to induce the tunableacoustic gradient (TAG) in the TAG lens may be under the control ofcomputer software; The mirrors that direct the laser excitation in thesingle beam incarnation and may act as an off/on switch for themulti-laser design may be controlled by computer software; The laserintensity via an attenuation wheel and tuning to different frequenciesmay be controlled by software input; Microscope stage movement may beunder software control; Movement of microscope objective or associatedfiber optics may be under software control; Edge finding, illumination,and control of the inverted objective by movement or on/off status maybe under software control; any imaging or light path controls (mirrors,shutters, scanning optics, SLMs, DMD etc.) may be under control ofsoftware.

To accommodate rapid printing, the objective 200 may be equipped with afiber optic cable. FIG. 6A illustrates an embodiment of a removable andattachable fiber optic cable accessory 250. In this embodiment, theaccessory 250 may comprise a fiber optic cable 252 and a fitting (notshown in FIGS. 6A-6B) which is attachable to the multi-photon tissueprinting print-head (not shown in 6A-6B). The fiber optic cable 252 canthen be positioned within the media 126 of the media chamber 122, asillustrated in FIG. 6B. Thus, the multi-photon laser beam 120 may passthrough the objective 200 and the fiber optic cable 252 to deliver thelaser energy to the media 126, creating the desired complex tissuestructure 260. To avoid moving the microscope objective during theprinting process or the printing vessel that contains delicate tissuestructures, the fiber optic cable itself may be moved if larger regionsof tissue need to be printed. In some cases, the accessory 250 can besterilized or replaced so that direct insertion into the media 126 doesnot compromise sterility or cross-contaminate printed cells.

Depending upon the power input into the fiber optic cable, multi-photonlasers may be capable of inducing irreversible damage to the core of thefiber optic cable. Thus, in some cases, induced wavelength chirping bygroup delayed dispersion (GDD) may be provided to minimize thispotential damage, by effectively dispersing the photons to elongate thelaser pulse. This may be used to either minimize damage to cells in theprint media or to extend the life of fiber optic cables. In suchinstances, a GDD device may be provided in the laser system 116 afterthe SLM or DMD and before entry to the print-head optics 118.

In some cases, three-dimensional printing of the desired tissue may becarried out with a single objective 200 or an objective 200 with anattached fiber optic accessory 250, wherein the one to three differentconfigurations, each associated with a distinct laser line andrepresenting a distinct shape or portion of the tissue may be pulsedthough the same objective 200. In such cases, a timed shutter system maybe installed such that there is no or minimal interference betweenimages being projected. Thus, laser multiplexing may be employed toallow generation of portions of the tissue structure simultaneously atmultiple points while utilizing the same CAD model of the tissuestructure. Likewise, the laser multiplexing may utilize different butcontiguous CAD based tissue models, minimizing the movement needed forlarger structure printing while decreasing overall print time further.For example, a vascular bed may have internal structures such as valvesin the larger blood vessels that prevent venous back flow in normalcirculation. These valve structures may be printed simultaneously withthe blood vessel walls. In such a case, the scaffolding associated withthe valve structure and/or blood vessel walls may be difficult to printseparately.

The instantaneously formed three-dimensional structure may be repeatedthroughout the print space during one round of printing. In biologicalsystems, small units may often be repeated throughout the structure.Therefore, repeated generation of a same structure in one print roundmay be useful for generating functional tissues. Additional,non-repetitive, fine featured structures and subsequent structures fromthe same cell-print material may be created that line-up with or link tothe first structure printed.

In some embodiments, the multi-photon tissue printing print-head 118 mayinclude multiple printing “heads” or sources of multi-photon excitationvia a first laser objective 200 a, a second laser objective 200 b, and athird laser objective 200 c as illustrated in FIGS. 7-8. FIG. 7illustrates an embodiment wherein the multi-photon tissue printingprint-head 118 may include a first laser objective 200 a, a second laserobjective 200 b, and a third laser objective 200 c, wherein the firstlaser objective 200 a may include a first fiber optic cable accessory250 a, the second laser objective 200 b may include a second fiber opticcable accessory 250 b, and the third laser objective 200 c may include athird fiber optic cable accessory 250 c. The first fiber optic cableaccessory 250 a, the second fiber optic cable accessory 250 b, and thethird fiber optic cable accessory 250 c may be directed into a singlemedia chamber 122. The media chamber 122 may have an open top or asealed top with port access by each accessory fiber optic cableaccessory (i.e., via the first fiber optic cable accessory 250 a, thesecond fiber optic cable accessory 250 b, and the third fiber opticcable accessory 250 c). This arrangement may increase the speed oflarge, rapid tissue printing, while maintaining control over the finaltissue structure. In some cases, the first fiber optic cable accessory250 a, the second fiber optic cable accessory 250 b, and the third fiberoptic cable accessory 250 c may deliver a projection of the same tissuestructure. In other cases, each the first fiber optic cable accessory250 a, the second fiber optic cable accessory 250 b, and the third fiberoptic cable accessory 250 c may deliver a first laser beam projection120 a, a second laser beam projection 120 b, and a third laser beamprojection 120 c, respectively, of a different tissue structure. Giventhe flexible arrangement of the multiple laser objectives and theability of directing the fiber optic cables into the same area withinthe media chamber 122, the tissue structures may be simultaneouslyprinted. The resulting tissue structures may be linked or not linkedtogether. The print time of a given tissue structure may have an inverserelationship to the number of laser delivery elements with someconsideration for the movement restrictions and considerations to beaccounted for with each additional excitation source.

FIG. 8 illustrates an embodiment wherein the multi-photon tissueprinting print-head 118 may include a first objective 200 a, a secondobjective 200 b, a third objective 200 c, a fourth objective 200 d, afifth objective 200 e, and a sixth objective 200 f, wherein eachobjective may include a first fiber optic cable accessory 250 a, asecond fiber optic cable accessory 250 b, a third fiber optic cableaccessory 250 c, a fourth fiber optic cable accessory 250 d, a fifthfiber optic cable accessory 250 e, and a sixth fiber optic cableaccessory 250 f, respectively, directed into a separate first mediachamber 122 a, a second media chamber 122 b, a third media chamber 122c, a fourth media chamber 122 d, a fifth media chamber 122 e, and asixth media chamber 122 f, respectively. The plurality of media chambersmay be a multi-well plate, wherein each well of the multi-well plate isa separate, individual media chamber. In some cases, the first fiberoptic cable accessory 250 a, the second fiber optic cable accessory 250b, the third fiber optic cable accessory 250 c, the fourth fiber opticcable accessory 250 d, the fifth fiber optic cable accessory 250 e, andthe sixth fiber optic cable accessory 250 f may deliver at least oneprojection of the same tissue structure. This provides multiple copiesof the tissue structure simultaneously. In other cases, the first fiberoptic cable accessory 250 a, the second fiber optic cable accessory 250b, the third fiber optic cable accessory 250 c, the fourth fiber opticcable accessory 250 d, the fifth fiber optic cable accessory 250 e, andthe sixth fiber optic cable accessory 250 f may deliver a firstmulti-photon laser beam projection 120 a, a second multi-photon laserbeam projection 120 b, and a third multi-photon laser beam projection120 c of a different tissue structure. In some cases, the print time maybe greatly reduced due to the ability of producing multiple copiessimultaneously.

In some embodiments, the multi-photon tissue printing print-head 118 mayinclude a serial array of objectives comprising a first objective 200 a,a second objective 200 b, and a third objective 200 c, as illustrated inFIG. 9. In this embodiment, each objective may be aligned with aseparate media chamber. For example, the first objective 200 a may bealigned with a first media chamber 122 a, the second objective 200 b maybe aligned with a second media chamber 122 b, the third objective 200 cmay be aligned with a third media chamber 122 c. In some instances, themultiple media chambers may be wells of a multi-well plate 300. In someembodiments, the first objective 200 a, the second objective 200 b, andthe third objective 200 c may deliver projection of the same tissuestructure. In other cases, the laser beam projections may differ perwell. The first objective 200 a, the second objective 200 b (not shownin FIG. 10), and the third objective 200 c (not shown in FIG. 10) may beprogrammed to move over the multi-well plate 300 in the x and ydirections, as illustrated in FIG. 10, to deliver the laser beamprojections into each well. Alternatively, it may be appreciated thatthe objectives may remain stationary while the multi-well plate 300moves in the x and y directions. Thus, for example, a serial arrayhaving three objectives can print tissue in a six well plate in twosteps: three tissue structures simultaneously and then three more tissuestructures simultaneously. It may be appreciated that plates having anynumber of wells may be used including, but not limited to at least about96 wells to about 394 wells, or more. The multi-well plate 300 maycomprise at least a first media chamber 122 a. The multi-well plate 300may comprise at least 1 well. The multi-well plate 300 may comprise atleast 4 wells. The multi-well plate 300 may comprise at least 6 wells.The multi-well plate 300 may comprise at least 8 wells. The multi-wellplate 300 may comprise at least 12 well. The multi-well plate 300 maycomprise at least 16 wells. The multi-well plate 300 may comprise atleast 24 wells. The multi-well plate 300 may comprise at least 48 wells.The multi-well plate 300 may comprise at least 96 wells. The multi-wellplate 300 may comprise at least 384 wells. The multi-well plate 300 maycomprise at least 1536 wells.

It may be appreciated that in the embodiments described herein, themicroscope stage may be able to move, the microscope head may be able tomove, and/or an associated fiber optic cable attached to the printingobjective may be able to move in order to print larger spaces.

3D Printing Methods

In an aspect, the present disclosure provides a method for printing athree-dimensional (3D) biological material. The x, y, and z dimensionsmay be simultaneously accessed while using the methods provided herein.The biological material may be a biologically functional tissue. Thebiological material may develop into a biologically functional tissue.The biological material may comprise a fully formed, a printedvasculature, a microvasculature, a porous network, a tube network,and/or a pore architecture which may provide delivery and/or diffusionof a sufficient concentration of nutrients and oxygen that may beconducive to prevent necrosis. The biological material may comprise aprinted lymphatic network, a lymphatic vasculature, and/or a lymphaticmicrovasculature which may allow for biological functions including, butnot limited to interstitial fluid homeostasis, regulation of the immunesystem, regulation of the circulatory system, regulation ofinflammation, and lipid absorptions. The biological material maycomprise cells arranged in a structure and/or architecture similar tothe native tissue that is trying to replicate; thus, allowing forbiological function similar to the biological function of the nativetissue. Printing of ultra-fine tissue structures such as, but notlimited to fine blood vessels such as capillaries, single cell layers oftissue, and layers of hard and/or soft tissues with mechanicalproperties of bone, cartilage, and/or tendons may be created.

The method may comprise receiving a computer model of the 3D biologicalmaterial in computer memory. The computer model may be a computer-aideddesign (CAD) model. The CAD model may be a 3D wireframe, a 3D solidmodel such as a parametric model and a direct or explicit model, and/ora freeform surface model. The CAD model may be generated by a computerafter a physical prototype is scanned and/or imaged using a device suchas a 3D scanner, a computer tomography (CT) scanning device, astructured-light 3D scanner, a modulated light 3D scanner, a laserscanner, a microscope, or a magnetic resonance imaging (MRI) device. Insome cases, the prototype image or scan is converted to a CAD model byusing an algorithm that converts the prototype image or scan into asurface model, a mesh model, or a volume model. The method may comprisereceiving computer model comprising a partial 3D structure and/or acomplete 3D structure of the 3D biological material.

Next, the method may comprise providing a media chamber comprising amedium comprising a plurality of cells and one or more polymerprecursors. The medium may comprise cell constituents (e.g.,organelles). The medium may further comprise glutathione or a functionalvariant (or derivative) thereof. The plurality of cells may comprisecells of at least two different types. The method may further comprisesubjecting at least a portion of the at least subset of the plurality ofcells to differentiation to form the cells of the at least two differenttypes. The at least the subset of the plurality of cells may comprisecells of at least two different types. The plurality of cells maycomprise the cells of the at least two different types.

The media chamber may be multi-well plate, a chamber slide, a tissueculture slide, a container, a flask, a bioreactor chamber, a vessel, abag such as a cell culture bag, a petri dish, a roller bottle, or acustom-fabricated well. The media chamber may be composed ofpolystyrene, glass, quartz, polypropylene, cyclo-olefin, or polyvinylchloride (PVC). The media chamber may be surface treated. Non-limitingexamples of surface treatments include plasma surface treatment, coatingwith carboxyl groups, hydroxyl groups, free amino groups, and/orpoly-D-lysine to promote cell attachment, and/or coating with ahydrophilic and neutrally charged hydrogel layer to inhibit cellattachment. The media chamber may comprise a volume ranging from e.g. atleast about 1 μl to about 30 L. The media chamber may comprise a volumeranging from e.g. at least about 1 μl to about 5 L. The media chambermay comprise a volume ranging from e.g. at least about 1 μl to about 1L. The media chamber may comprise a volume ranging from e.g. at leastabout 1 μl to about 0.5 L. The media chamber may comprise a volumeranging from e.g. at least about 1 μl to about 250 ml. The media chambermay comprise a volume ranging from e.g. at least about 1 μl to about 100ml. The media chamber may comprise a volume ranging from e.g. at leastabout 1 μl to about 50 ml. The media chamber may comprise a volumeranging from e.g. at least about 1 μl to about 25 ml. The media chambermay comprise a volume ranging from e.g. at least about 1 μl to about 10ml. The media chamber may comprise a volume ranging from e.g. at leastabout 1 μl to about 5 ml. The media chamber may comprise a volumeranging from e.g. at least about 1 μl to about 1 ml. The media chambermay comprise a volume ranging from e.g. at least about 1 μl to about 500μl. The media chamber may comprise a volume ranging from e.g. at leastabout 1 μl to about 100 μl. The media chamber may comprise a volumeranging from e.g. at least about 1 μl to about 50 μl. The media chambermay comprise a volume ranging from e.g. at least about 1 μl to about 25μl. The media chamber may comprise a volume ranging from e.g. at leastabout 1 μl to about 10 μl. The media chamber may comprise a volumeranging from e.g. at least about 1 μl to about 5 μl.

The media chamber may comprise a volume of about 1 μl. The media chambermay comprise a volume of about 10 μl. The media chamber may comprise avolume of about 100 μl. The media chamber may comprise a volume of about1000 μl. The media chamber may comprise a volume of about 5 ml. Themedia chamber may comprise a volume of about 10 ml. The media chambermay comprise a volume of about 20 ml. The media chamber may comprise avolume of about 30 ml. The media chamber may comprise a volume of about40 ml. The media chamber may comprise a volume of about 50 ml. The mediachamber may comprise a volume of about 60 ml. The media chamber maycomprise a volume of about 70 ml. The media chamber may comprise avolume of about 5 ml. The media chamber may comprise a volume of about80 ml. The media chamber may comprise a volume of about 90 ml. The mediachamber may comprise a volume of about 100 ml. The media chamber maycomprise a volume of about 200 ml. The media chamber may comprise avolume of about 300 ml. The media chamber may comprise a volume of about400 ml. The media chamber may comprise a volume of about 500 ml. Themedia chamber may comprise a volume of about 600 ml. The media chambermay comprise a volume of about 700 ml. The media chamber may comprise avolume of about 800 ml. The media chamber may comprise a volume of about900 ml. The media chamber may comprise a volume of about 1000 ml. Themedia chamber may comprise a volume of about 2 L. The media chamber maycomprise a volume of about 3 L. The media chamber may comprise a volumeof about 4 L. The media chamber may comprise a volume of about 5 L. Themedia chamber may comprise a volume of about 6 L. The media chamber maycomprise a volume of about 7 L. The media chamber may comprise a volumeof about 8 L. The media chamber may comprise a volume of about 9 L. Themedia chamber may comprise a volume of about 10 L. The media chamber maycomprise a volume of about 20 L. The media chamber may comprise a volumeof about 30 L. The media chamber may comprise a volume of about 40 L.The media chamber may comprise a volume of about 50 L. The media chambermay comprise a volume of about 60 L. The media chamber may comprise avolume of about 70 L. The media chamber may comprise a volume of about80 L. The media chamber may comprise a volume of about 90 L. The mediachamber may comprise a volume of about 100 L or more.

The medium may comprise a plurality of cells, one or more polymerprecursors, cell constituents (e.g., organelles), and/or cell culturemedium. The polymer precursors may comprise at least two differentpolymeric precursors. The polymeric precursors may be a polymerizablematerial. The polymer precursors may comprise collagen. Non-limitingexamples of collagen types in the medium include fibrillar collagen suchas type I, II, III, V, and XI collagen, fibril associated collagens withinterrupted triple helices (FACIT) collagen such as type IX, XII, andXIV collagen, short chain collagen such as type VIII and X collagen,basement membrane collagen such as type IV collagen, type VI collagen,type VII collagen, type XIII collagen, or any combination thereof. Thepolymer precursors may comprise extracellular matrix componentsincluding, but not limited to proteoglycans such as heparan sulfate,chondroitin sulfate, and keratan sulfate, non-proteoglycanpolysaccharide such as hyaluronic acid, and elastin, fibronectin,laminin, nidogen, or any combination thereof. In some instances, thepolymer precursors may comprise polyglycolic acid (PGA), polylactic acid(PLA), alginate, polyethylene oxide, polyethylene glycol,polypropyleneoxide, poly(N-isopropylacrylamide), chitosan, fibrin,fibrinogen, polylactic acid-polyglycolic acid (PLGA) copolymer,poly(methyl methacrylate) (PMMA), polyvinyl alcohol (PVA),poly(propylene fumarate)s (PPFs), polycaprolactone (PCL), poly(β-aminoester), gelatin, dextran, chondroitin sulfate, or any combinationthereof. Non-limiting examples of cell culture medium include Dulbecco'sModified Eagle Medium (DMEM), serum-free cell culture medium, RPMI 1640medium, Minimum Essential Media (MEM), Iscove's Modified Dulbecco'sMedium (IMDM), and Opti-MEM™ I Reduced Serum Medium.

The medium may further comprise a plurality of beads. In some cases, theat least portion of the 3D biological material, as formed, may includethe plurality of beads. The at least portion of the 3D biologicalmaterial, as formed, may include the plurality of microspheres and/orparticles. The beads, microspheres, and/or particles may range in sizefrom about 1 nanometer to about 200 micrometers. The beads,microspheres, and/or particles may be chemically inert. The beads,microspheres, and/or particles may be hollow. The beads, microspheres,and/or particles may be solid. The beads, microspheres, and/or particlesmay comprise a core and a shell. The beads, microspheres, and/orparticles may be polymeric, magnetic, porous, metallic, fluorescent,dyed, hydrogel, lipid, or any combination thereof. The beads,microspheres, and/or particles may comprise latex, at least one type ofextracellular matrix protein, a cell, a drug, a biopolymer, a lipid, abiocompatible polymer, a small molecule, or any combination thereof.Non-limiting examples of biopolymers include fibrin, fibrinogen,chitosan, cellulose, dextran, chitin, gelatin, collagen, glycogen,starch, and lignin. Non-limiting examples of biocompatible polymersinclude collagen, hyaluronic acid, chondroitin sulfate, polyglycolicacid (PGA), polylactic acid, alginate, polyethylene oxide (PEO),polyethylene glycol (PEG), polypropyleneoxide,poly(N-isopropylacrylamide), chitosan, fibrin, polylactic-co-glycolicacid (PLGA) copolymer, or any combinations thereof.

The beads may further comprise signaling molecules or proteins. Thesignaling molecules or proteins may promote formation of the 3Dbiological material to permit organ function. The beads, microspheres,and/or particles may be functionalized with a protein, nucleic acid,and/or dye. The beads, microspheres, and/or particles may befunctionalized with streptavidin. The surface of the beads,microspheres, and/or particles may be coated with at least one signalingmolecule, a protein such as an antibody, a nucleic acid such as a DNAand/or RNA molecule, a polymer, a small molecule, and/or a dye. Thebeads, microspheres, and/or particles may encapsulate a payload such as,for example, a cell, a drug, a signaling molecule, a protein, a nucleicacid, a small molecule, a dye, and/or a polymer such as a biopolymer.The biodegradable beads, microspheres, and/or particles may havecontrolled release of the payload. The beads, microspheres, and/orparticles may be biodegradable. The biodegradable beads, microspheres,and/or particles may have a controlled and/or customizable degradationrate. The beads, microspheres, and/or particles may benon-biodegradable. The signaling molecules, proteins, nucleic acids,and/or any other material that is comprised by the beads, microspheres,and/or particles may promote formation of the 3D biological material topermit organ function. Non-limiting examples of the signaling molecules,small molecules, and proteins, such as antibodies, that may haveagonist, antagonist, growth, and/or cell differentiating activitiesinclude: transforming growth factor-β (TGF-β), vascular endothelialgrowth factor (VEGF), fibroblast growth factors (FGFs) such as FGF-1 andFGF-2, platelet-derived growth factor (PDGF), angiopoietin-1 (Ang1),Ang2, matrix metalloproteinases (MMPs), delta-like ligand 4 (Dll4),class 3 semaphorins, macrophage colony-stimulating factor (M-CSF),granulocyte-macrophage colony-stimulating factor (M-CSF), bonemorphogenic protein 4 (BMP4), transforming growth factor (TGF), ActivinA, retinoic acid (RA), epidermal growth factor (EGF), thiazovivin.

Next, the method may comprise directing at least one energy beam to themedium in the media chamber along at least one energy beam path that ispatterned into a 3D projection in accordance with computer instructionsfor printing the 3D biological material in computer memory, to form atleast a portion of the 3D biological material. The portion of the 3Dbiological material may comprise at least a subset of the plurality ofcells and a polymer formed from the one or more polymer precursors. Theat least portion of the 3D biological material may comprisemicrovasculature for providing one or more nutrients to the plurality ofcells. The microvasculature may be a blood microvasculature, a lymphaticmicrovasculature, or any combination thereof. The microvasculature mayhave a cross-section from about 1 micrometer (μm) to about 20 μm. The 3Dbiological material may have a thickness or diameter from about 100 μmto about 5 centimeters (cm).

The method may comprise directing at least one energy beam to the mediumin the media chamber along at least one energy beam path in accordancewith the point-cloud representation or lines-based representation of thecomputer model of the 3D biological material, to subject at least aportion of the polymer precursors to form at least a portion of the 3Dbiological material. The method may comprise directing at least oneenergy beam to the medium in the media chamber along at least one energybeam path in accordance with a computer model of a partial 3D structureand/or a complete 3D structure of the 3D biological material.

The method may further comprise directing at least one energy beam tothe medium the media chamber along one or more additional energy beampaths to form at least another portion of the 3D biological material.The method may further comprise directing at least two energy beams tothe medium in the media chamber along at least two energy beam paths inaccordance with the computer instructions, to permit multiple portionsof the medium in the media chamber to simultaneously form at least aportion of the 3D biological material. The at least two energy beams maybe of identical wavelengths. The at least two energy beams may be ofdifferent wavelengths.

The computer instructions may comprise a set of images corresponding tothe 3D biological material. The computer instructions may directadjustment of at least one or more parameters of the at least one energybeam as a function of time during formation of the 3D biologicalmaterial, and/or location of a stage upon which the 3D biologicalmaterial is formed.

The at least another portion of the 3D biological material may be linkedto the 3D biological material formed by directing at least one energybeam to the medium the media chamber along at least one energy beampath. The at least another portion of the 3D biological material may belinked to the 3D biological material formed by directing at least oneenergy beam to the medium the media chamber along one or more additionalenergy beam paths. The at least another portion of the 3D biologicalmaterial may not be linked to the 3D biological material formed bydirecting at least one energy beam to the medium the media chamber alongat least one energy beam path. The at least another portion of the 3Dbiological material may not be linked to the 3D biological materialformed in by directing at least one energy beam to the medium the mediachamber along one or more additional energy beam paths.

The energy beam may be a multi-photon laser beam 120. The at least oneenergy beam may comprise coherent light. The at least one energy beammay be generated by a laser. The at least one energy beam may be phasemodulated. The energy beam may be polarized and/or re-combined withother beams. As an alternative, the energy beam may be non-coherentlight. The at least one energy beam may be a multi-photon energy beam.The multi-photon energy beam may be a two-photon energy beam. The energybeam source (e.g., laser) may provide energy (e.g., laser beam) having awavelength from e.g. about 300 nm to about 5 mm. The energy beam (e.g.,laser) may provide energy (e.g., laser beam) having a wavelength fromabout e.g. 350 nm to about 1800 nm. The energy beam (e.g., laser) mayprovide energy (e.g., laser beam) having a wavelength from e.g. about1800 nm to about 5 mm. The energy beam (e.g., laser) may provide energy(e.g., laser beam) having a wavelength of about 300 nm. The energy beam(e.g., laser) may provide energy (e.g., laser beam) having a wavelengthof about 400 nm. The energy beam (e.g., laser) may provide energy (e.g.,laser beam) having a wavelength of about 600 nm. The energy beam (e.g.,laser) may provide energy (e.g., laser beam) having a wavelength ofabout 700 nm. The energy beam (e.g., laser) may provide energy (e.g.,laser beam) having a wavelength of about 800 nm. The energy beam (e.g.,laser) may provide energy (e.g., laser beam) having a wavelength ofabout 900 nm. The energy beam (e.g., laser) may provide energy (e.g.,laser beam) having a wavelength of about 1000 nm. The energy beam (e.g.,laser) may provide energy (e.g., laser beam) having a wavelength ofabout nm. The energy beam (e.g., laser) may provide energy (e.g., laserbeam) having a wavelength of about 1200 nm. The energy beam (e.g.,laser) may provide energy (e.g., laser beam) having a wavelength ofabout 1300 nm. The energy beam (e.g., laser) may provide energy (e.g.,laser beam) having a wavelength of about 1400 nm. The energy beam (e.g.,laser) may provide energy (e.g., laser beam) having a wavelength ofabout 1500 nm. The energy beam (e.g., laser) may provide energy (e.g.,laser beam) having a wavelength of about 1600 nm. The energy beam (e.g.,laser) may provide energy (e.g., laser beam) having a wavelength ofabout 1700 nm. The energy beam (e.g., laser) may provide energy (e.g.,laser beam) having a wavelength of about 1800 nm. The energy beam (e.g.,laser) may provide energy (e.g., laser beam) having a wavelength ofabout 1900 nm. The energy beam (e.g., laser) may provide energy (e.g.,laser beam) having a wavelength of about 2000 nm. The energy beam (e.g.,laser) may provide energy (e.g., laser beam) having a wavelength ofabout 3000 nm. The energy beam (e.g., laser) may provide energy (e.g.,laser beam) having a wavelength of about 4000 nm. The energy beam (e.g.,laser) may provide energy (e.g., laser beam) having a wavelength ofabout 5000 nm.

Next, the method may comprise generating a point-cloud representation orlines-based representation of the computer model of the 3D biologicalmaterial in computer memory. The method may generate suchrepresentations prior to directing at least one energy beam to themedium in the media chamber. The method may use the point-cloudrepresentation or the lines-based representation to generate thecomputer instructions. The point-cloud representation or the lines-basedrepresentation may comprise multi-dimensional structural elementscorresponding to the 3D biological material. The point-cloudrepresentation or the lines-based representation may comprise structuralelements in two dimensions. The point-cloud representation or thelines-based representation may comprise structural elements in threedimensions. The point-cloud representation or the lines-basedrepresentation may comprise elements in both two dimensions and threedimensions. The structural elements may be associated with tissuefunction and/or cellular segregation. The structural elements may be avessel, a lymph vessel, a vasculature, a microvasculature, a muscle, aligament, a tendon, a bone matrix, a cartilage matrix, a connectivetissue matrix, an extracellular matrix, a nerve network, a scaffold, orany combination thereof. The structural element may be a collagen fiber,a reticular fiber, an elastic fiber, a nerve fiber, a polymer fiber, achannel, a micro-channel, or any combination thereof.

A point-cloud representation is a set of data points defined in the x,y, and z planes by x, y, z coordinates that represent the externalsurface of an object (i.e., a prototype). A point-cloud representationmay be generated by a 3D modeling program to produce CAD files, or anyline drawing set. In some examples (e.g., printing a plastic or metalpart), a 3D scanner may be used to generate a 3D model of the object.Non-limiting examples of 3D scanning technologies include lasertriangulation 3D scanning, structured light 3D scanning, photogrammetry,contact based 3D scanning, and laser pulse or time of flight 3Dscanning. Laser triangulation 3D scanning technology involves projectinga laser beam on the surface of an object and measuring the deformationof the laser ray via a sensor. Based on the deformation of the laser rayand trigonometric triangulation, the laser triangulation systemcalculates a specific deviation angle. The calculated deviation angle isdirectly linked to the distance from the object to the scanner. When thelaser triangulation 3D scanner collects enough distances, it is capableof mapping the object's surface and to create a 3D scan. Structuredlight 3D scanning technology measures the deformation of a light patternon the surface of an object to generate a 3D scan of the surface of anobject. Structured light 3D scanning also uses trigonometrictriangulation, but relies on the projection of a series of linearpatterns instead of the projection of a laser beam. The structured light3D scanning system is then capable to examine the edges of each line inthe pattern and to calculate the distance from the scanner to theobject's surface. Photogrammetry, also called 3D scan from photography,reconstructs in 3D an object captured in a 2D image using computationalgeometry algorithms. The principle of photogrammetry is to analyzeseveral photographs of a static object, taken from different viewpoints,and to automatically detect pixels corresponding to a same physicalpoint. The data inputs required from the user are the parameters of thecamera such as focal length and lens distortion. The computationalgeometry algorithms then calculate the distances between coordinates andoutput a 3D image reconstruction of the object. Contact based 3Dscanning or digitizing, may rely on the sampling of several points onthe surface of an object, measured by the deformation of a probe.Contact 3D scanners probe the object through physical touch, while theobject is firmly held in place. A touching probe is moved on the surfaceof the object to record 3D information. The probe is sometimes attachedto an articulated arm capable of collecting all its respectiveconfigurations and angles for more precision. Laser pulse-based 3Dscanning, or time of flight 3D scanning, may be based on the time offlight of a laser beam. In laser pulse-based 3D scanning, the laser beamis projected on a surface and collected by a sensor. The time of travelof the laser beam between its emission and reception provides thegeometrical information of the object's surface.

Methods and systems of the present disclosure may be implemented by wayof at least one or more algorithms. An algorithm may be implemented byway of software upon execution by the central processing unit 1105. Thealgorithm may, for example, create a hologram or a holographic imagebased on a computer model. The algorithm may create a partial hologram.Pulse shaping of light may be achieved across the one or more SLMs byapplying the Gerchberg-Saxton algorithm or weighted Gerchberg-Saxtonalgorithm to create binary holographic images of structural elementsthat may then be projected to recreate the image in one, two, and/orthree dimensions. Other algorithms that may be useful in wavefrontshaping include, but are not limited to Lohmann, Lohmann type III, andmixed-region amplitude freedom (MRAF) algorithms. Additionalpre-processing and post-processing of the images may occur toaccommodate different types of desired structural prints, differentprint media responses to the incoming light pulses, and any limitationin the optical system or cell viability or changes in projectionsystems. These changes in data processing may include, but are notlimited to Fourier Transforms, selective masks resulting in pixelremoval, and/or overlaying holograms for printing in different planessimultaneously to produce the 3D hologram. In addition, these processesmay require additional slicing or redistribution of the holographicdata.

A line-based representation of the computer model of the 3D biologicalmaterial may be defined as a collection of lines, vertices, edges,surfaces, dots or collections of linked dots of various sizes, and/orfaces that define the shape of the 3D biological material. The faces maycomprise triangles (triangle meshes), quadrilaterals, convex polygons,concave polygons, and/or polygons with holes. Non-limiting examples oflines-based representations of the computer model of the 3D biologicalmaterial in computer memory may include 3D line drawings, 2D linedrawings, polygon meshes, and freeform surface models. A polygon mesh isa collection of vertices, edges and faces that defines the shape of anobject in 3D computer modeling. A freeform surface model describes thesurface of a 3D object. A freeform surface model may be created byconstruction of curves from which the 3D surface is then swept throughand by the manipulation of the surface via poles or control points thatdefine the shape of the surface.

After generating a point-cloud representation or lines-basedrepresentation of the computer model of the 3D biological material, themethod may further comprise converting the point-cloud representation orlines-based representation into an image. The image may be a hologram ora holographic image. The image may be a partial hologram. The image maybe projected in a holographic manner. The image may be projected as apartial hologram. The image may be deconstructed and reconstructed priorto projection in a holographic manner.

The method may further comprise providing yet another media chamber,comprising a medium comprising a plurality of cells comprising cells ofat least two different types and one or more polymer precursors, alongone or more additional energy beam paths to form at least anotherportion of the 3D biological material. The other portion of the 3Dbiological material may be linked to the first 3D biological materialformed. The other portion of the 3D biological material may bechemically cross-linked to the first 3D biological material formed. Theother portion of the 3D biological material may be mechanically linkedto the first 3D biological material formed. Non-limiting examples ofmechanical coupling includes joints, hinges, locking joints and hinges,Velcro-like elements, springs, coils, points of stretch, interlockingloops, sockets, gears, ratchets, screws, and chain links. The otherportion of the 3D biological material may be cohesively coupled to thefirst 3D biological material formed. The other portion of the 3Dbiological material may be linked to the first 3D biological materialvia printing and active deposition of structure using 3D holographicprinting, cells, extracellular matrix deposited by the cells, and/or apre-existing structure formed by other non-biological approaches. Theother portion of the 3D biological material may be polymerized to thefirst 3D biological material formed. The other portion of the 3Dbiological material may not be linked to the first 3D biologicalmaterial formed.

The method may comprise direct linking of the 3D biological materialwith other tissue, which linking may be in accordance with a computermodel. The method may comprise directly linking, merging, bonding, orwelding 3D printed material with already printed structures, wherelinking is in accordance with a computer model. In some cases, themethod may provide linking of the 3D biological material with othertissue, which may involve chemical cross-linking, mechanical linking,and/or cohesively coupling.

The method may further comprise directing at least two energy beams tothe medium in the media chamber along at least two energy beam paths inaccordance with the computer model of the 3D biological material, topermit multiple portions of the medium in the media chamber tosimultaneously form at least a portion of the 3D biological material.The at least two energy beams may be of identical wavelengths. The twoenergy beams may be of different wavelengths.

The portion of the 3D biological material may comprise microvasculaturefor providing one or more nutrients to the plurality of cells. Themicrovasculature may be a blood microvasculature, a lymphaticmicrovasculature, or any combination thereof. The microvasculature mayhave a cross-section e.g. from about 1 μm to about 20 μm. Thecross-section may be e.g. from about 1 μm to about 10 μm. The 3Dbiological material may have a thickness or diameter e.g. from about 100μm to about 5 centimeter (cm). The 3D biological material may have athickness or diameter e.g. from about 200 μm to about 3 cm. The 3Dbiological material may have a thickness or diameter e.g. from about 300μm to about 1 cm.

The 3D biological material may have a thickness or diameter e.g. fromabout 0.1 μm to about 10 cm. The 3D biological material may have athickness or diameter e.g. from about 0.1 μm to about 5 cm. The 3Dbiological material may have a thickness or diameter e.g. from about 0.1μm to about 4 cm. The 3D biological material may have a thickness ordiameter e.g. from about 0.1 μm to about 3 cm. The 3D biologicalmaterial may have a thickness or diameter e.g. from about 0.1 μm toabout 2 cm. The 3D biological material may have a thickness or diametere.g. from about 0.1 μm to about 1 cm.

The 3D biological material may have a thickness or diameter e.g. fromabout 0.1 μm to about 9 mm. The 3D biological material may have athickness or diameter e.g. from about 0.1 μm to about 8 mm. The 3Dbiological material may have a thickness or diameter e.g. from about 0.1μm to about 7 mm. The 3D biological material may have a thickness ordiameter e.g. from about 0.1 μm to about 6 mm. The 3D biologicalmaterial may have a thickness or diameter e.g. from about 0.1 μm toabout 5 mm. The 3D biological material may have a thickness or diametere.g. from about 0.1 μm to about 4 mm. The 3D biological material mayhave a thickness or diameter e.g. from about 0.1 μm to about 3 mm. The3D biological material may have a thickness or diameter e.g. from about0.1 μm to about 2 mm. The 3D biological material may have a thickness ordiameter e.g. from about 0.1 μm to about 1 mm. The 3D biologicalmaterial may have a thickness or diameter e.g. from about 0.1 μm toabout 0.5 mm. The 3D biological material may have a thickness ordiameter e.g. from about 0.1 μm to about 0.1 mm.

The 3D biological material may have a thickness or diameter e.g. fromabout 0.1 μm to about 90 μm. The 3D biological material may have athickness or diameter e.g. from about 0.1 μm to about 80 μm. The 3Dbiological material may have a thickness or diameter e.g. from about 0.1μm to about 70 μm. The 3D biological material may have a thickness ordiameter e.g. from about 0.1 μm to about 60 μm. The 3D biologicalmaterial may have a thickness or diameter e.g. from about 0.1 μm toabout 50 μm. The 3D biological material may have a thickness or diametere.g. from about 0.1 μm to about 40 μm. The 3D biological material mayhave a thickness or diameter e.g. from about 0.1 μm to about 30 μm. The3D biological material may have a thickness or diameter e.g. from about0.1 μm to about 20 μm. The 3D biological material may have a thicknessor diameter e.g. from about 0.1 μm to about 10 μm. The 3D biologicalmaterial may have a thickness or diameter e.g. from about 0.1 μm toabout 5 μm. The 3D biological material may have a thickness or diametere.g. from about 0.1 μm to about 4 μm. The 3D biological material mayhave a thickness or diameter e.g. from about 0.1 μm to about 3 μm. The3D biological material may have a thickness or diameter e.g. from about0.1 μm to about 2 μm. The 3D biological material may have a thickness ordiameter e.g. from about 0.1 μm to about 1 μm. The 3D biologicalmaterial may have a thickness or diameter e.g. from about 0.1 μm toabout 0.75 μm. The 3D biological material may have a thickness ordiameter e.g. from about 0.1 μm to about 0.5 μm. The 3D biologicalmaterial may have a thickness or diameter e.g. from about 0.1 μm toabout 0.25 μm.

The 3D biological material may be printed in a time period of e.g. atleast about 0.01 hour to about 700 hours. The 3D biological material maybe printed in a time period of e.g. at least about 0.01 hour to about600 hours. The 3D biological material may be printed in a time period ofe.g. at least about 0.01 hour to about 500 hours. The 3D biologicalmaterial may be printed in a time period of e.g. at least about 0.01hour to about 400 hours. The 3D biological material may be printed in atime period of e.g. at least about 0.01 hour to about 350 hours. The 3Dbiological material may be printed in a time period of e.g. at leastabout 0.01 hour to about 300 hours. The 3D biological material may beprinted in a time period of e.g. at least about 0.01 hour to about 250hours. The 3D biological material may be printed in a time period ofe.g. at least about 0.01 hour to about 200 hours. The 3D biologicalmaterial may be printed in a time period of e.g. at least about 0.01hour to about 150 hours. The 3D biological material may be printed in atime period of e.g. at least about 0.01 hour to about 100 hours. The 3Dbiological material may be printed in a time period of e.g. at leastabout 0.01 hour to about 72 hours. The 3D biological material may beprinted in a time period of e.g. at least about 0.01 hour to about 48hours. The 3D biological material may be printed in a time period ofe.g. at least about 0.01 hour to about 36 hours. The 3D biologicalmaterial may be printed in a time period of e.g. at least about 0.01hour to about 24 hours. The 3D biological material may be printed in atime period of e.g. at least about 0.01 hour to about 12 hours. The 3Dbiological material may be printed in a time period of e.g. at leastabout 0.01 hour to about 6 hours. The 3D biological material may beprinted in a time period of e.g. at least about 0.01 hour to about 3hours. The 3D biological material may be printed in a time period ofe.g. at least about 0.01 hour to about 2 hours. The 3D biologicalmaterial may be printed in a time period of e.g. at least about 0.01hour to about 1 hour. The 3D biological material may be printed in atime period of e.g. at least about 0.01 hour to about 0.5 hours.

The 3D biological material (or object) may be printed in a time periodof at most about 350 hours. The 3D biological material may be printed ina time period of at most about 300 hours. The 3D biological material maybe printed in a time period of at most about 250 hours. The 3Dbiological material may be printed in a time period of at most about 200hours. The 3D biological material may be printed in a time period of atmost about 150 hours. The 3D biological material may be printed in atime period of at most about 100 hours. The 3D biological material maybe printed in a time period of at most about 72 hours. The 3D biologicalmaterial may be printed in a time period of at most about 48 hours. The3D biological material may be printed in a time period of at most about36 hours. The 3D biological material may be printed in a time period ofat most about 24 hours. The 3D biological material may be printed in atime period of at most about 12 hours. The 3D biological material may beprinted in a time period of at most about 6 hours. The 3D biologicalmaterial may be printed in a time period of at most about 2 hours. The3D biological material may be printed in a time period of at most about1 hour.

The at least portion of the 3D biological material may comprise acell-containing scaffold. The cell-containing scaffold may comprise atleast a subset of the plurality of cells. The 3D biological material maycomprise a cell-containing scaffold, which cell-containing scaffold maycomprise at least a subset of the plurality of cells. The 3D biologicalmaterial, as formed, may include a plurality of cell-containingscaffolds. The 3D biological material may comprise cell-containingscaffolds. The plurality of cell-containing scaffolds may be coupledtogether. The cell-containing scaffolds may be cohesively ormechanically coupled together. The cell-containing scaffold may beempty, porous, and/or hollow. The cell-containing scaffold may serve asa complete element or portion of a supporting structure, collectingduct, or vascular element. The plurality of cell-containing scaffoldsmay be cohesively coupled together. The plurality of the cell-containingscaffolds may be mechanically coupled together. The plurality ofcell-containing scaffolds may be mechanically coupled together throughone or more members selected from the group consisting of joints,hinges, locking joints and hinges, Velcro-like elements, springs, coils,points of stretch, interlocking loops, sockets, gears, ratchets, screw,and chain links.

The cell-containing scaffolds may comprise a network. The network maycomprise a plurality of strands. The plurality of strands may form amesh structure. The plurality of strands may form a grid structure. Theplurality of strands may form a sheet structure. The plurality ofstrands may form a tube structure. The plurality of strands may form apore network. The plurality of strands may form a cylindrical structure.The plurality of strands may form a rectangular structure. The pluralityof strands may form a square structure. The plurality of strands mayform a tiered or layered structure. The plurality of strands may form alattice structure. The plurality of strands may form a porous structure.The plurality of strands may form a net-like structure. The plurality ofstrands may form an interconnected structure. The plurality of strandsmay form a channeled structure. The plurality of strands may form ahexagonal structure. The plurality of strands may form a cagedstructure. The plurality of strands may form a sphere. The plurality ofstrands may form a polygon.

The individual strands of the plurality of strands may have a thicknessfrom about 0.1 nanometers (nm) to about 5 cm. The plurality of strandsmay have a thickness e.g. from about 0.1 nm to about 10 cm. Theplurality of strands may have a thickness e.g. from about 0.1 nm toabout 5 cm. The plurality of strands may have a thickness e.g. fromabout 0.1 nm to about 4 cm. The plurality of strands may have athickness e.g. from about 0.1 nm to about 3 cm. The plurality of strandsmay have a thickness e.g. from about 0.1 nm to about 2 cm. The pluralityof strands may have a thickness e.g. from about 0.1 nm to about 1 cm.The plurality of strands may have a thickness e.g. from about 0.1 nm toabout 0.5 cm.

The plurality of strands may have a thickness e.g. from about 0.1 nm toabout 1000 μm. The plurality of strands may have a thickness e.g. fromabout 0.1 nm to about 900 μm. The plurality of strands may have athickness e.g. from about 0.1 nm to about 800 μm. The plurality ofstrands may have a thickness e.g. from about 0.1 nm to about 700 μm. Theplurality of strands may have a thickness e.g. from about 0.1 nm toabout 600 μm. The plurality of strands may have a thickness e.g. fromabout 0.1 nm to about 500 μm. The plurality of strands may have athickness e.g. from about 0.1 nm to about 400 μm. The plurality ofstrands may have a thickness e.g. from about 0.1 nm to about 300 μm. Theplurality of strands may have a thickness e.g. from about 0.1 nm toabout 200 μm. The plurality of strands may have a thickness e.g. fromabout 0.1 nm to about 100 μm. The plurality of strands may have athickness e.g. from about 0.1 nm to about 50 μm. The plurality ofstrands may have a thickness e.g. from about 0.1 nm to about 25 μm. Theplurality of strands may have a thickness e.g. from about 0.1 nm toabout 10 μm. The plurality of strands may have a thickness e.g. fromabout 0.1 nm to about 1 μm.

The plurality of strands may have a thickness e.g. from about 0.1 nm toabout 900 nm. The plurality of strands may have a thickness e.g. fromabout 0.1 nm to about 800 nm. The plurality of strands may have athickness e.g. from about 0.1 nm to about 700 nm. The plurality ofstrands may have a thickness e.g. from about 0.1 nm to about 600 nm. Theplurality of strands may have a thickness e.g. from about 0.1 nm toabout 500 nm. The plurality of strands may have a thickness e.g. fromabout 0.1 nm to about 400 nm. The plurality of strands may have athickness e.g. from about 0.1 nm to about 300 nm. The plurality ofstrands may have a thickness e.g. from about 0.1 nm to about 200 nm. Theplurality of strands may have a thickness e.g. from about 0.1 nm toabout 100 nm. The plurality of strands may have a thickness e.g. fromabout 0.1 nm to about 50 nm. The plurality of strands may have athickness e.g. from about 0.1 nm to about 25 nm. The plurality ofstrands may have a thickness e.g. from about 0.1 nm to about 10 nm. Theplurality of strands may have a thickness e.g. from about 0.1 nm toabout 1 nm. The plurality of strands may have a thickness e.g. fromabout 0.1 nm to about 0.5 nm. The plurality of strands may have athickness e.g. from about 0.1 nm to about 0.25 nm.

The plurality of strands may have a thickness e.g. from about 0.1 nm toabout 800 μm. The plurality of strands may have a thickness e.g. fromabout 0.1 μm to about 1 μm. The plurality of strands may have e.g. athickness from about 1 μm to about 100 μm. The plurality of strands mayhave a thickness e.g. from about 1 millimeter (mm) to about 100 mm. Theplurality of strands may have a thickness e.g. from about 1 cm to about5 cm.

The method may comprise at least another portion of the 3D biologicalmaterial that may be formed within the at least portion of the 3Dbiological material.

The method may comprise forming another portion of the 3D biologicalmaterial within the portion of the 3D biological material that is firstformed, after first directing the at least one energy beam to the mediumin the media chamber. The method may comprise printing a 3D objectinside a previously printed 3D object. The method may comprise printinga 3D biological material inside a previously printed 3D object. Thepreviously printed structure may be a 3D object that may be formed of apolymeric material, a metal, metal alloy, composite material, or anycombination thereof. The 3D object may be formed of a polymericmaterial, in some cases including biological material (e.g., one or morecells or cellular components). Printing a 3D object inside a previouslyprinted 3D object can be possible with precision printing and energybeam excitation that is exact in the x, y, and z planes.

The method may comprise printing a 3D object inside a previously printed3D structure by using at least one energy beam having a wavelength inthe near-infrared light spectrum. Near-infrared wavelengths canpenetrate tissue and structures. The method may comprise directing theat least one energy beam having a wavelength in the near-infrared lightspectrum into the medium as a 3D projection corresponding to a 3D objectmay allow the 3D projection to penetrate a previously printed 3D object.The method may comprise directing the at least one energy beam having awavelength in the near-infrared light spectrum into the medium as a 3Dprojection corresponding to a 3D biological material may allow the 3Dprojection to penetrate a previously printed 3D biological material. The3D projection may be a hologram. The 3D projection may be a partialhologram. The energy beam having a wavelength in the near-infrared lightspectrum can have minimal or no scattering. When directed as a 3Dprojection into the medium, the energy beam having a wavelength in thenear-infrared light spectrum can coalesce as a hologram inside a tissueor a previously printed 3D object, structure, and/or biologicalmaterial.

The near-infrared light spectrum can range from about 650 nanometers(nm) to 1 millimeters (mm). Within the near-infrared light spectrum, thenear-infrared (NIR) window (i.e., optical window or therapeutic window)defines the range of wavelengths from e.g. about 650 to 1350 nm wherelight has its maximum depth of penetration in tissue. Furthermore, thefar-red light spectrum can range from about 710 nm to about 850 nm. Theenergy beam used in the methods disclosed herein can be an NIR energybeam having a wavelength in the NIR light spectrum. The energy beam usedin the methods disclosed herein can be an NIR energy beam having awavelength in the NIR window light spectrum. The energy beam used in themethods disclosed herein can be an NIR energy beam having a wavelengthin the far-red light spectrum.

The NIR energy beam (e.g., laser) may provide energy (e.g., laser beam)having a wavelength ranging from about 650 nm to about 1 mm. The NIRenergy beam (e.g., laser) may provide energy (e.g., laser beam) having awavelength ranging from about 710 nm to about 850 nm or more. The NIRenergy beam (e.g., laser) may provide energy (e.g., laser beam) having awavelength ranging from about 650 nm to about 1350 nm or more. The NIRenergy beam (e.g., laser) may provide energy (e.g., laser beam) having awavelength of about 650 nm. The NIR energy beam (e.g., laser) mayprovide energy (e.g., laser beam) having a wavelength of about 700 nm.The NIR energy beam (e.g., laser) may provide energy (e.g., laser beam)having a wavelength of about 710 nm. The NIR energy beam (e.g., laser)may provide energy (e.g., laser beam) having a wavelength of about 750nm. The NIR energy beam (e.g., laser) may provide energy (e.g., laserbeam) having a wavelength of about 800 nm. The NIR energy beam (e.g.,laser) may provide energy (e.g., laser beam) having a wavelength ofabout 850 nm. The NIR energy beam (e.g., laser) may provide energy(e.g., laser beam) having a wavelength of about 900 nm. The NIR energybeam (e.g., laser) may provide energy (e.g., laser beam) having awavelength of about 950 nm. The NIR energy beam (e.g., laser) mayprovide energy (e.g., laser beam) having a wavelength of about 1000 nm.The NIR energy beam (e.g., laser) may provide energy (e.g., laser beam)having a wavelength of about 1200 nm. The NIR energy beam (e.g., laser)may provide energy (e.g., laser beam) having a wavelength of about 1300nm. The NIR energy beam (e.g., laser) may provide energy (e.g., laserbeam) having a wavelength of about 1350 nm. The NIR energy beam (e.g.,laser) may provide energy (e.g., laser beam) having a wavelength ofabout 1500 nm. The NIR energy beam (e.g., laser) may provide energy(e.g., laser beam) having a wavelength of about 2000 nm. The NIR energybeam (e.g., laser) may provide energy (e.g., laser beam) having awavelength of about 2500 nm. The NIR energy beam (e.g., laser) mayprovide energy (e.g., laser beam) having a wavelength of about 3000 nm.The NIR energy beam (e.g., laser) may provide energy (e.g., laser beam)having a wavelength of about 3500 nm. The NIR energy beam (e.g., laser)may provide energy (e.g., laser beam) having a wavelength of about 4000nm. The NIR energy beam (e.g., laser) may provide energy (e.g., laserbeam) having a wavelength of about 5000 nm.

The energy beam having a wavelength in the NIR light spectrum may bedirected to the medium in the media chamber along at least one energybeam path in accordance with a point-cloud representation, lines-basedrepresentation, partial 3D structure, complete 3D structure, or a 3Dprojection (i.e., hologram or partial hologram) of the 3D biologicalmaterial. In some examples, a partial or complete 3D structured isprojected into the medium at the same time. The energy beam having awavelength in the NIR light spectrum may penetrate previously formedstructures within the media chamber. The energy beam having a wavelengthin the NIR light spectrum may penetrate previously formedcell-containing scaffolds within the media chamber. The energy beamhaving a wavelength in the NIR light spectrum may penetrate previouslyformed 3D biological material located within the media chamber. Theenergy beam having a wavelength in the NIR light spectrum that isdirected to the medium in the media chamber may subject at least aportion of the polymer precursors to form at least a portion of the 3Dbiological material within a previously formed portion of the 3Dbiological material. The energy beam having a wavelength in the NIRlight spectrum that is directed to the medium in the media chamber maysubject at least a portion of the polymer precursors to form at least aportion of the 3D object within a previously formed portion of the 3Dobject. The specific NIR wavelengths of the energy beam used in themethods provided herein can enable the printing of 3D biologicalmaterials within previously formed structures by penetrating thepreviously formed structures at least one energy beam path in accordancewith the 3D projection (i.e., hologram or partial hologram) of the 3Dbiological material.

In another aspect, the present disclosure provides an additional methodof printing a three-dimensional (3D) biological material. The method maycomprise receiving a computer model of the 3D biological material incomputer memory.

Next, the method may comprise generating a point-cloud representation orlines-based representation of the computer model of the 3D biologicalmaterial in computer memory.

Next, the method may provide a media chamber comprising a first medium,wherein the first medium comprises a first plurality of cells and afirst polymeric precursor. The first medium may comprise glutathione ora functional variant (or derivative) thereof.

Next, the method may comprise directing at least one energy beam to thefirst medium in the media chamber along at least one energy beam path inaccordance with computer instructions for printing the 3D biologicalmaterial in computer memory, to subject at least a portion of the firstmedium in the media chamber to form a first portion of the 3D biologicalmaterial.

Next, the method may comprise providing a second medium in the mediachamber, wherein the second medium comprises a second plurality of cellsand a second polymeric precursor, wherein the second plurality of cellsis of a different type than the first plurality of cells. The secondmedium may comprise glutathione or a functional variant (or derivative)thereof.

Next, the method may comprise directing at least one energy beam to thesecond medium in the media chamber along at least one energy beam pathin accordance with the computer instructions, to subject at least aportion of the second medium in the media chamber to form at least asecond portion of the 3D biological material.

The method may further comprise removing a remainder of the first mediumfrom the media chamber to leave the first portion of the d 3D biologicalmaterial in the media chamber. The first portion of the 3D biologicalmaterial left in the medium chamber may be removably fixed to the mediachamber.

The method may further comprise generating a point-cloud representationor lines-based representation of the 3D biological material in computermemory. The method may further use the point-cloud representation orlines-based representation to generate the computer instructions. Themethod may further comprise converting the point-cloud representation orlines-based representation into an image or image set that may be usedto spatially modulate an incoming coherent light source such thatbiological structures may be projected in one dimension. The method mayfurther comprise converting the point-cloud representation orlines-based representation into an image or image set that may be usedto spatially modulate an incoming coherent light source such thatbiological structures may be projected in two dimensions. The method mayfurther comprise converting the point-cloud representation orlines-based representation into an image or image set that may be usedto spatially modulate an incoming coherent light source such thatbiological structures may be projected in three dimensions. The methodmay further comprise converting the point-cloud representation orlines-based representation into an image or image set that may be usedto spatially modulate at least one incoming coherent light source suchthat biological structures may be projected in a mixture ofone-dimensional, two-dimensional and/or three-dimensional structures.The method image or image set may be projected in a holographic manner.The image or image set may be deconstructed and reconstructed intopartial elements or representative structures prior to projection in aholographic manner. The point-cloud representation or the lines-basedrepresentation may comprise multi-dimensional structural elementscorresponding to the 3D biological material. The point-cloudrepresentation or the lines-based representation may comprise structuralelements in two dimensions. The structural elements may be associatedwith tissue function and/or cellular segregation. The point-cloudrepresentation or the lines-based representation may comprise structuralelements in three dimensions, wherein the structural elements may beassociated with tissue function and/or cellular segregation.

The at least one energy beam may comprise coherent light. The at leastone energy beam may be generated by a laser. The at least one energybeam may be phase modulated. The at least one energy beam may be phasemodulated and raster-scanned throughout the sample medium. The at leasta portion of the 3D biological material may comprise microvasculaturefor providing one or more nutrients to the plurality of cells. Themedium may further comprise a plurality of beads. The at least portionof the 3D biological material, as formed, may include the plurality ofbeads. The 3D biological material may be printed in a time period of atmost about 350 hours. The 3D biological material may be printed in atime period of at most about 72 hours. 3D biological material may beprinted in a time period of at most about 12 hours. The method maycomprise at least another portion of the 3D biological material that maybe formed within the first portion of the 3D biological material and/orthe second portion of the 3D biological material.

A variety of tissue structures can be generated with the rapidmulti-photon printing system 100 such as thick, complex tissue layerswhich include blood vessels, lymphatic vasculature, interstitial cellnetworks, cell niches or a-cellular elastic structures, to name a few.In many instances, the three-dimensional projection from computergenerated three dimensional models may be created from scans or maps ofnative tissue structures which allows for precise replication of nativetissues. Such tissues may be comprised of a variety of different celltypes, each organizing in a specific manner so as to generate a specificstructure or provide a particular function. For example, blood vessels,such as arteries and veins, may be comprised of endothelial cells, basallamina (a layer of extracellular matrix), connective tissue and layersof smooth muscle cells. A tissue containing blood vessels may alsoinclude cells forming the tissue surrounding the blood vessels. Forexample, liver tissue may also include hepatocytes. Hepatocytes may begrouped in the liver into similar functional units and are similar inappearance, but they may express different genes depending on theirlocation. This compartmentalization may allow the liver to carry out themultiple functions of the liver in different locations. Every cell maynot participate in the oxidation of proteins, detoxification of reactiveoxygen species, and bile productions. These tasks may be given todifferent groups of hepatocytes depending on their location in theliver. The rest of the cells in the liver (collectively callednon-parenchymal cells) may be found in compartments between the massivenumbers of hepatocytes. Thus, when printing liver tissue, the cell typesrelated to the formation of blood vessels may be appropriately arrangedso as to promote and support the organization of these cells into bloodvessels; and the hepatocytes and non-parenchymal cells may be likewiseappropriately arranged to form the desired end result tissue. Sucharrangement may be achieved by printing layers of nets and otherstructures within the tissue for temporary cell organization in threedimensions. The structure of most organs may require multiple cell typesto be layered and grouped as functional units wherein some cells supportthe function of these units as described above and some are actualfunctional elements. Organs may be vascularized to maintain cellularhealth within these functional units that comprise the entire organ. Inthe case of immune system function, the highly organized structure ofthe lymph node may facilitate the ability of the cells to properlyrespond to infectious agents. To properly respond to an infection,multiple cell-types may come into contact with each other to exchangeinformation through cell-surface contact about the pathogen or agentthat is eliciting the immune response. These contacts may beorchestrated by release of cell-signaling molecules and have patternsand contact timing. Disruption of cell-cell interactions ordisorganization of a lymphatic tissue, wherein cells are scattered ornot in their normal area within the tissue, may be causal or associatedwith inability for an immune system to respond properly or develophighly selected for antibodies. Therefore, reconstruction of tissuessuch as this for the purpose of transplant or drug development maybenefit not only from the placement of microvasculature which nourishesand supports cells, but the placement of the cells themselves such thatthey can be organized to interact and produce the necessary signals toexecute tissue function, as is necessary within the lymph node duringresponse to an infectious agent.

In an aspect, the present disclosure provides a method for printing athree-dimensional (3D) object. The method may comprise directing atleast one energy beam into a medium comprising one or more precursors togenerate the 3D object. The 3D object may comprise a material formedfrom the one or more precursors. The one or more precursors may bepolymeric precursors. The one or more precursors may include one or moremetals. The one or more precursors may include glass or sand precursors.The one or more precursors may be a powder. The material may be apolymeric material. The material may include at least one metal. Thematerial may include glass or sand (e.g. green sand). The material mayinclude a mixture of a polymeric precursor, a metallic precursor, and/ora glass precursor. For example, the material may be alumide (i.e., amixture of polyamide and aluminum), a mixture of polyamide and glass,and/or a mixture of nylon and glass. The polymeric material may be apowder. The polymeric material may be contained in a fabrication powderbed. Non-limiting examples of polymeric materials may include nylon,polystyrene, polyamide, polyethylene, polystyrene, polyether etherketone (PEEK), polypropylene, polybutylene terephthalate, thermoplasticpolyurethane, thermoplastic elastomer, and polyoxomethylene. The nylonmaterial may be glass-filled nylon, fiber-filled nylon, or durablenylon. The polyamide may be flame-retardant polyamide. Non-limitingexamples of metals may include steel, titanium, metal alloy mixtures,and aluminum. The metal alloy mixtures may include nickel chromium andcobalt chrome alloys.

Next, the at least one energy beam may be directed into the medium as a3D projection. The 3D projection may correspond to the 3D object. The 3Dprojection may be a hologram. The 3D projection may be a partialhologram. The 3D projection may be a holographic image. The hologram orholographic image may be a one-dimensional, two-dimensional, and/orthree-dimensional image. The method may comprise receiving a computermodel of the 3D object in computer memory. The computer model may be acomputer-aided design (CAD) model. The CAD model may be a 3D wireframe,a 3D solid model such as a parametric model and a direct or explicitmodel, and/or a freeform surface model. The CAD model may be generatedby a computer after a physical prototype is scanned and/or imaged usinga device such as a 3D scanner, a computer tomography (CT) scanningdevice, a structured-light 3D scanner, a modulated light 3D scanner, alaser scanner, a microscope, or a magnetic resonance imaging (MRI)device. In some cases, the prototype image or scan is converted to a CADmodel by using an algorithm that converts the prototype image or scaninto a surface model, a mesh model, or a volume model. The method maycomprise receiving a computer model comprising a partial 3D structureand/or a complete 3D structure of the 3D object. The systems disclosedherein used for printing 3D biological materials may be the same as thesystems used for printing 3D objects.

The medium may comprise cells or cellular constituents. The cellularconstituents may include, but are not limited to organelles such asmitochondria, nuclei, ribosomes, vesicles, Golgi apparatuses,cytoskeleton components, smooth endoplasmic reticulum, vacuoles, andchloroplasts; phospholipids; and cellular membranes.

In an aspect, the present disclosure provides a method for printing athree-dimensional (3D) biological material. The method may comprisedirecting at least a first energy beam into a media chamber comprising afirst medium. The first medium may comprise a first plurality of cellsand a first polymeric precursor to generate a first portion of the 3Dbiological material. The method may comprise directing at least a secondenergy beam into the media chamber comprising a second medium. Thesecond medium may comprise a second plurality of cells and a secondpolymeric precursor, to generate a second portion of the 3D biologicalmaterial. The second portion of the 3D biological material may beadjacent to the first portion of the 3D biological material.

The at least first energy beam and the at least second energy beam maybe from the same energy source. The at least first energy beam and theat least second energy beam may be laser beams. The cells of the firstplurality of cells and the cells of the second plurality of cells may beof different types. The cells of the first plurality of cells and thecells of the second plurality of cells may be of the same type. Thefirst polymeric precursor and the second polymeric precursor may bedifferent. The first polymeric precursor and the second polymericprecursor may be the same.

Net Creation

Nets may be created within the media 126 by polymerizing thepolymerizable material in a pattern and manner so as to create a desirednet structure 500 amongst the cells. FIG. 11 illustrates an example of anet structure 500 formed from polymerizable material. In this example,the net structure 500 may have a grid shape formed from net strands 502.The net strands 502 may have a thickness between e.g. at least about0.0001 micrometers to about 100 micrometers. FIG. 12A illustrates a netstructure 500 comprised of strands 502 having a thickness ofapproximately 0.1 micrometers and FIG. 12B illustrates a net structure500 comprised of strands 502 having a thickness of at least about 5micrometers. Different sized strands may be used for creating meshnetworks of different sizes and densities to promote cell-cellcommunication, allowing cell movement to be promoted or prevented, orsupporting tissue properties such that there may be differences inelasticity, strength, or compression forces associated with differentmesh net structures. As shown in FIG. 11, in some cases, the netstructure 500 may have apertures 504 that are sized to allow specificcells 506 to pass through and restrict the passage of other cells. Insome cases, apertures 504 may range in size e.g. from at least about 3micrometers to about 100 micrometers. FIG. 11 illustrates a netstructure 500 having apertures 504 sized and configured so as to preventpassage of any cell 506 which is at least about 4 micrometers in size(such as e.g. about 4 micrometers to about 100 micrometers)therethrough. This may temporarily trap cells 506 within the netstructure 500 as the net structure 500 is generated. It may beappreciated that the net structures may have various apertures 504 ofany geometric shape, such as round, hexagonal, octagonal or square.

In some cases, these cell-size specific nets may be designed to isolaterounded cells of specific types. Rounding of cells may be induced bychemical changes to the environment or temperature changes, and acombination of these may be used during the printing process. Roundedcells in certain physiologic conditions may not move or crawl, but maybe suspended in place. FIG. 13 illustrates rounded cells 506 temporarilytrapped within a net structure 500. In this example, the apertures 504may be smaller than the rounded diameter of the cell type when suspendedin media 126, but larger than the estimated diameter of the cellnucleus. In some instances, the apertures 504 may be the same size as orabout 1 micrometer smaller than the cell nucleus. These sizes may beselected to create temporary confinement of the particular cells 506.Cells may pass through an aperture that is larger than the cell nucleusbut may be confined by one that is smaller. In this embodiment, the net500 may be printed under conditions which cause the cells 506 to berounded so as to be trapped by the net 500. Once the printed materialsare returned to physiologic conditions, the cells 506 may no longerround and may be able to crawl and move through the apertures 504, asillustrated in FIG. 14. FIG. 14 shows a first net structure 500 a and asecond net structure 500 b disposed near each other so that cells 506may be able to move through the apertures 504 and engage underphysiological conditions allowing cell-cell contact, reordering andnatural proliferation while maintaining gross structural arrangement andsupport. Together, the cell layers and niches created by the first netstructure 500 a and the second net structure 500 b may form asupra-structure of cell-containing elements designed to facilitatecell-cell contact and movement during three-dimensional tissuedevelopment in culture.

The first net structure 500 a and the second net structure 500 b maythen be disposed of, reabsorbed, degraded or otherwise lost by the finalstages of tissue development. In some instances, the first net structure500 a and the second net structure 500 b may be lost by enzymaticdigestion by cells expressing matrix metalloproteinases, or othermethods.

Net Features

The nets may include a variety of features to assist in the creation ofvarious types of tissues and tissue structures. Such features mayinclude, but are not limited to variations in thickness, density, andstructural design to influence the movement of cells within the netstructure 500 and/or to affect the overall shape of the net 500.Additional features may include, but are not limited to variousmechanical elements to assist in shaping the overall net 500, such aslinking portions of the net to itself or other nets, and to furtherinfluence the form of the final tissue structure. These and other netfeatures are described herein.

In some cases, the nets may be created with various structural featuresby changing the intensity, prolonging the exposure or repeating exposureof the multi-photon laser beam 120 projected at various sites within themedia 126. In some instances, intensity changes or prolonged exposure atcertain critical points in the media 126 may create features in the netstructure that may influence the density of cells deposited. This canlead to mechanical differences at these points which can be used intissue construction. FIGS. 15A-15C illustrate a method of creating areasof such structural features within a net structure 500. FIG. 15A showsthe generation of a net structure 500 by projecting the multi-photonlaser beam 120 from the optics of the multi-photon tissue printingprint-head 118 into the media 126. FIG. 15B illustrates a secondprojection of a multi-photon laser beam 120′ from the laser beamtargeting specific coordinates 520 within the net structure 500. In thisembodiment, the specific coordinates 520 may coincide withpre-determined intersections of the net strands 502 of the net structure500. The second projection 120′ may be at the same or differingwavelength from the first projection 150. This second projection of amulti-photon laser beam 120′ may increase the density of net material atthe specific coordinates 520. FIG. 15C illustrates the final netstructure 500 having the various points of reinforcement at thepre-determined intersections of the net strands 502.

FIGS. 16A-16C illustrate another method of creating areas of suchstructural features within a net structure 500. FIG. 16A shows thegeneration of a net structure 500 by projecting the multi-photon laserbeam 120 from the optics of the multi-photon tissue printing print-head118 into the media 126. FIG. 16B illustrates a second projection 120′ ofa multi-photon laser beam targeting specific coordinates 520 within thenet structure 500. In this embodiment, the specific coordinates maycoincide with various net strands which together form a structuralfeature 530 having a zig-zag shape. The second projection of amulti-photon laser beam 120′ may be at the same or differing wavelengthfrom the first projection of a multi-photon laser beam 120. This secondprojection of a multi-photon laser beam 120′ may increase the density ofnet material at the specific coordinates 520. FIG. 16C illustrates thefinal net structure 500 having the reinforced zig-zag shaped structuralfeature 530. Similar to parallel tube support and re-enforcements oflinear capillaries, many tubes have branches that may be supported. Thezigzag shape of reinforcement for tissues and cell nets can be used, inone example, in parallel printed reinforcements to support branchedcapillary structures in printed tissues. In another embodiment, layersof a zig-zag shape may provide structural support in response toperpendicular compression forces and parallel shear forces.

Having lines or regions of high density within cell nets, allows forcells to deform tissues along certain guidelines. One such demonstrationof a structural use may be the organization of fibroblasts to formtissues around vascular epithelial cells meant to form blood vessels. Asimple sheet of fibroblasts may result in deformation that does notsupport the tube structure of a capillary and thus compromising thefunction of and structure of printed capillary structures. Instead,thicker net regions such as parallel line reinforcement can directstructural deformation in a manner that is supportive of the desiredtissue structure such as a tube of vascular endothelial cells, asillustrated in FIG. 16D.

In some embodiments, increased areas of thickness along a net structure500 are used to influence cells to engage in high-tension interactions.Such interactions may cause the overall net structure 500 to form foldsor wrinkles which may be desirable for the ultimate tissue structure.FIGS. 17A-17B and 18A-18B illustrate the use of structural featureswithin a net to cause the tissue structure to fold or wrinkle in aparticular manner as a result of cell-cell contact and movement duringthree-dimensional tissue development in culture. FIG. 17A illustrates anet structure 500 formed within the media 126 wherein the net structure500 includes structural reinforcements 540 along particular net strandsand a first unreinforced portion 503 a and a second unreinforced portion503 b of the net therebetween. In some embodiments, such structuralreinforcements 540 are made by constructing the net structure 500 withnet strands of different diameters. In the embodiment of FIG. 17A, afirst net strand 502 a and a second net strand 502 b have a largerdiameter than other strands within the net structure 500 and arearranged in parallel to each other separated by a distance. The largerdiameter serves as reinforcement. A third net strand 502 c has a smallerdiameter than the first net strand 502 a and the second net strand 502 band is arranged in parallel to the first net strand 502 a and the secondnet strand 502 b located therebetween. The third net strand 502 c isalso reinforced to a lesser degree. The remaining net portions areunreinforced and reside between the first net strand 502 a, the secondnet strand 502 b, and the third net strand 502 c that are reinforced, asshown. Cells 506, such as fibroblasts, are trapped within the netstructure 500, amongst the first unreinforced portion 503 a and a secondunreinforced portion 503 b of the net structure 500, between the firstnet strand 502 a, the second net strand 502 b, and the third net strand502 c that are reinforced. The cells 506 then begin the process of cellinteraction and cell movement. Since the cells 506 can freelycommunicate within the first unreinforced portion 503 a and a secondunreinforced portion 503 b of the net structure 500, tension forcesresult from cell-cell interactions. This draws the first net strand 502a and the second net strand 502 b toward each other, as illustrated inFIG. 17B. The third net strand 502 c keeps the first unreinforcedportion 503 a and a second unreinforced portion 503 b separated whichbegin to fold or wrinkle. Since the third net strand 502 c is reinforcedto a lesser degree, the cells 506 along the wrinkles are able tointeract over and around the third net strand 502 c, further stabilizingthe folded shape. FIGS. 18A-18B illustrate the net structure 500embodiment of FIGS. 17A-17B from a side view. FIG. 18A illustrates thedownward motion of the cells 506 (indicated by arrows 532) as the cellsmove and communicate, to form the folds. FIG. 18B illustrates the cells506 having formed the folds between the first net strand 502 a, thesecond net strand 502 b, and the third net strand 502 c drawing thefirst net strand 502 a and the second net strand 502 b toward eachother.

FIGS. 19A-19C illustrate another embodiment of a net structure 500having increased areas of thickness to influence or force cells toengage in high-tension interactions, leading to folds or wrinkles. FIG.19A illustrates a net structure 500 formed within media 126 wherein thenet structure 500 includes a first structural reinforcement 540 a, asecond structural reinforcement 540 b, and a third structuralreinforcement 540 c along particular net strands and unreinforcedportions of the net therebetween. More specifically, the firststructural reinforcement 540 a, the second structural reinforcement 540b, and the third structural reinforcement 540 c have the shape of linesor elongate areas positioned in a parallel manner so that portions ofthe first unreinforced portion 503 a and the second unreinforced portion503 b of the net structure 500 reside therebetween. Cells 506, such asfibroblasts, are trapped within the net structure 500, amongst the firstunreinforced portion 503 a and the second unreinforced portion 503 b andbetween the first structural reinforcement 540 a, the second structuralreinforcement 540 b, and the third structural reinforcement 540 c. Thecells 506 then begin the process of cell interaction and cell movement.Since the cells 506 can freely communicate within the first unreinforcedportion 503 a and the second unreinforced portion 503 b of the netstructure 500, tension forces result from cell-cell interactions. Thisdraws the first net strand 502 a and the second net strand 502 b towardeach other, as illustrated in FIG. 19B. The reinforcements keep thefirst unreinforced portion 503 a and the second unreinforced portion 503b separated which begin to fold or wrinkle. FIG. 19C provides a sideview of the tissue showing the first unreinforced portion 503 a, thesecond unreinforced portion 503 b, and the third unreinforced portion503 cb drawing together, forming folds or wrinkles.

In some embodiments, variations in density of the net structure 500guide movement and interactions of cells 506. FIG. 20 illustrates anexample net structure 500 having a high density net region 560surrounded by a low density net region 562. The high density net region560 is comprised of apertures that are smaller than the apertures of thelow density net region 562. Therefore, the high density region 560 has ahigher number of apertures compared to the low density net region 562.The small aperture size of the high density net region 560 resistsmovement of cells 506 there through. Thus, when the cells 506 move andinteract, the cells 506 avoid the high density net region 560, creatinga tissue structure around or surrounding the high density net region560. Thus, once the net structure 500 dissolves, degrades, or isotherwise removed, a hole or passageway remains in the place of the highdensity net region 560. When multiple nets are layered so that the highdensity net regions are aligned, a lumen may be formed through the bodyof surrounding cells 506. This is one way in which a low density regionof cells while concentrating cells in other areas may be done in asingle printed structure.

In some cases, the high density net region 560 may comprise a signalingmolecule, a cytokine, a protein, a surface coating, a polymer such as ahydrophilic polymer, and/or a surface treatment such as plasmatreatment, that inhibits cell migration, adhesion, and/or traction. Insome cases, the low density net region 562 may comprise a signalingmolecule, a cytokine, a protein, a surface coating, a polymer such as ahydrophobic polymer, and/or a surface treatment, that promotes cellmigration, adhesion, and/or traction.

FIG. 21 illustrates another embodiment wherein variations in density ofthe net structure 500 guide movement and interactions of cells 506. Inthis embodiment, the net structure 500 comprises a first net portion 570and a second net portion 572, wherein a high density net region 574resides therebetween. In addition, a ladder 576 is formed of a lowerdensity net region which extends through the high density net region560, bridging the first net portion 570 and the second net portion 572.Thus, cells 506 trapped in the first net portion 570 and/or the secondnet portion 572 are able to move along the ladder 576 while avoiding thehigh density net regions 560. This guides cells 506 in a predetermineddirection and allows the cells 506 to form tissue structures accordingto predetermined shapes. It may be appreciated that in other embodimentsthe high density net region 560 is absent, wherein no netting materialis present. This also guides cells 506 along the ladder 576,particularly when the ladder 576 comprises features which promote celladhesion or attraction.

FIG. 22 illustrates another embodiment wherein variations in density ofthe net structure 500 guide movement and interactions of cells 506 tomake a three-dimensional tissue structure. In this embodiment, the netstructure 500 comprises a first net portion 570 and a second net portion572, wherein a high density net region 560 resides therebetween. Thus,cells 506 trapped in the first net portion 570 and/or the second netportion 572 are unable to move due to the high density net regions 560.This guides cells 506 in a predetermined direction and allows the cells506 to form tissue structures according to predetermined shapes.

FIGS. 23A-23E, 24A-24B, 25 illustrate textured elements 600 along netstrands 502 which promote cell adhesion, attraction, and/or interaction.Textured elements may be constructed with divots, raised notches, roughedges, or any element that purposely creates a surface that is notperfectly smooth for the purpose of cell adhesion and/or cellinteraction with the surface.

The cell nets may be formed with specific enzyme cleavage sites as partof the natural structural material such that the native activity ofmatrix metalloproteinases may be encouraged to remodel printedstructures to allow for cell movement, flow, and/or cell-cellinteractions. A non-limiting example list of such enzyme cleavage siteswithin a protein based structure are given in Table 2.

TABLE 2 Examples of enzyme cleavage sites within a protein-basedstructure. Cell express example Enzyme Substrate example Fibroblast MMP1Collagen Epithelial cell MMP9 Gelatin, Collagen Macrophage MMP12 Elastin

FIG. 26 illustrates an embodiment of a net structure 500 having acleavage site 610. Thus, the net structure 500 includes uncleavablepolymer strands and cleavable polymer strands. In this embodiment, thenet structure 500 includes a fibroblast activation protein (FAP) whichallows cells 506 to pass therethrough. Monomers that are polymerized inthe printing process to create cell containing biogels may alsoincorporate proteins that have matrix metalloproteinase (MMP) cleavagesites to allow cells to engage in functional remolding of depositedstructures. MMP responsive proteins that may be incorporated in theprint media or used to polymerize into specific structures include butare not limited to proteins; collagens I, II, III, VII, VIII, X,gelatin, fibronectin, and elastin.

In some embodiments, the nets include printed mechanical elements whichare designed to provide specific functions within a tissue structure orto assist in joining various tissue structures together. Such mechanicalelements include joints, hinges, locking joints and hinges, Velcro-likeelements, springs, coils, points of stretch, interlocking loops,sockets, gears, ratchets, screws, and chain links, to name a few. Themechanical elements may be printed so as to be embedded within a net,disposed along a surface of a net (such as along a flat surface or alongan edge), or in a location so as to assist in joining or linking twoportions of the net together or two separate nets together. Thus, insome embodiments, a layered tissue structure is formed by linkingtogether individual nets with the use of mechanical elements. In otherembodiments, an unlinked structural niche is embedded within printedvascular networks. When the niche is printed as an unlinked proximalstructure, or a new structure with links attached to structures printedpreviously, these cell containing nets form semi-autonomous, activestructures composed of moving cells, and additional elements designed tofacilitate cell-cell contact and movement during tissue development inculture.

It may be appreciated that many of the mechanical elements are comprisedof individual portions that are mateable together, such as two joinableportions of a hinge or two interlocking loops. In such embodiments, theportions of the mechanical element may be printed in a matedconfiguration. In other embodiments, the portions may be mated afterprinting as sheets or edges with mateable units may be brought intoclose proximity during movement of tissues in response to celldevelopment and exerted forces therein or in response to external forcessuch as pressures along airways or vasculature. In some embodiments, themechanical element is printed so that a first portion is attached to afirst net and a second portion is attached to a second net. Upon mating,the first and second nets are able to move in relation to each other atthe location of the mechanical element. This may assist in joiningvarious net structures together to create a complex three-dimensionaltissue structure, particularly in a manner which benefits fromrelational movement in the development process. It may be appreciatedthat the mechanical may alternatively or additionally be printed so thatthe first portion is attached to a first portion of a net and the secondportion is attached to a second portion of the same net, wherein theportions of the net move in relation to each other at the location ofthe mechanical element. This may assist in wrapping, looping, twistingor other desired movement within a net during the development process.

FIGS. 27A-27B illustrate an embodiment of a mechanical elementcomprising a pivot joint 700. In this embodiment, as shown in FIG. 27A,the pivot joint 700 comprises a first protrusion 702 having a first head704 with a rounded surface 705 and a second protrusion 706 having asecond head 708 with a concave surface 709. The concave surface 709 ismateable with the rounded surface 705 so that the first head 704 is ableto pivot against the concave surface 709 in a single direction, such asin a rocking motion. In some embodiments, the joint 700 is printed sothat the first protrusion 702 is attached to a first net structure 500 aand the second protrusion 706 is attached to a second net structure 500b, as illustrated in FIG. 27B. Upon mating, the first net structure 500a and the second net structure 500 b are able to pivot in relation toeach other at the location of the pivot joint 700.

FIGS. 28A-28B illustrate an embodiment of a mechanical elementcomprising a ball-and-socket joint 720. In this embodiment, as shown asa cross-sectional view in FIG. 28A, the ball-and-socket joint 720comprises a first protrusion 702 having a rounded ball head 724 and asecond protrusion 706 having a concave socket head 728. The concavesocket head 728 is mateable with the rounded ball head 724 so that therounded ball head 724 is able to rotate within the concave socket head728 in a manner similar to an anatomical ball-and-socket joint. In someembodiments, the ball-and-socket joint 720 is printed so that the firstprotrusion 702 is attached to a first net structure 500 a and the secondprotrusion 706 is attached to a second net structure 500 b, asillustrated in FIG. 28B. Upon mating, the first net structure 500 a andthe second net structure 500 b are able to rotate in relation to eachother, in numerous directions, at the location of the ball-and-socketjoint 720.

FIGS. 29A-29B illustrate an embodiment of a mechanical elementcomprising a saddle joint 740. In this embodiment, as shown in FIG. 29A,the saddle joint 740 comprises a first protrusion 702 having a firsthead 704 with a saddle-shaped indentation 745 and a second protrusion706 having a second head 708 with a corresponding second saddle-shapedindentation 749. The first head 704 and the second head 706 may beoriented in a 90 degree offset so that the first indentation 745 and thesecond indentation 749 are mateable as illustrated. Thus, the first head704 and the second head 706 are able to rotate around each other in asingle direction. In some embodiments, the saddle joint 740 is printedso that the first protrusion 702 is attached to a first net structure500 a and the second protrusion 706 is attached to a second netstructure 500 b, as illustrated in FIG. 29B. Upon mating, the first netstructure 500 a and the second net structure 500 b are able to pivot inrelation to each other, in a single direction, at the location of thesaddle joint 740.

FIGS. 30-31 illustrate an embodiment of a mechanical element comprisinga socket joint 760. In this embodiment, as shown in FIG. 30, the socketjoint 760 comprises a first protrusion 702 having a first head 704 witha socket-shaped cavity 765 and a second protrusion 706 having a secondhead 708 shaped to fit within the socket-shaped cavity 765. In thisembodiment, the socket-shaped cavity 765 is tubular in shape and thesecond head 708 is cylindrical in shape so as to be insertable into thesocket-shaped cavity 765. The second head 708 is able to slidelongitudinally and rotate within the socket-shaped cavity 765. In someembodiments, the socket joint 760 is printed so that the firstprotrusion 702 is attached to a first net structure 500 a and the secondprotrusion 706 is attached to a second net structure 500 b, asillustrated in FIG. 31. Upon mating, the first net structure 500 a andthe second net structure 500 b are able to slide and rotate in relationto each other, at the location of the socket joint 760.

FIGS. 32-33 illustrate an embodiment of a mechanical element comprisinga threaded joint 770. In this embodiment, as shown in FIG. 32, thethreaded joint 770 comprises a first protrusion 702 having a first head704 with a socket-shaped cavity 765 having a first groove 777 a, asecond groove 777 b, and a third groove 777 c and a second protrusion706 having a second head 708 with a first thread 779 a, a second thread779 b, and a third thread 779 c. Wherein the second head 708 is shapedto fit within the socket-shaped cavity 765 so that the first thread 779a, the second thread 779 b, and the third thread 779 c mate with thefirst groove 777 a, the second groove 777 b, and the third groove 777 cin a screw type manner. In some embodiments, the threaded joint 770 isprinted so that the first protrusion 702 is attached to a first netstructure 500 a and the second protrusion 706 is attached to a secondnet structure 500 b, as illustrated in FIG. 33. Upon mating, the firstnet structure 500 a and the second net structure 500 b are able torotate in relation to each other, at the location of the threaded joint770, with a resistance to longitudinal sliding due to the threads.

FIGS. 34A-34B illustrate an embodiment of a mechanical elementcomprising a coil or spring 800. In this embodiment, as shown in FIG.34A, the spring 800 has a first end 802, a second end 804 and a coiledor spiral configuration therebetween so as to provide spring tensionbetween the first end 802 and the second end 804. In some embodiments,the spring 800 is printed so that the first end 802 is attached to afirst net structure 500 a and the second end 804 is attached to a secondnet structure 500 b, as illustrated in FIG. 34B. Thus, the first netstructure 500 a and the second net structure 500 b are able to move inrelation to each other while the spring 800 maintains connection.

FIGS. 35A-35B illustrate an embodiment of a mechanical elementcomprising a chain 810. In this embodiment, as shown in FIG. 35A, thechain 810 has a first end 802, a second end 804 and a first link 816 a,a second link 816 b, a third link 816 c, and a fourth link 816 dtherebetween in a chain configuration so as to connect the first end 802and the second end 804 together. In some embodiments, the chain 810 isprinted so that the first end 802 is attached to a first net structure500 a and the second end 804 is attached to a second net structure 500b, as illustrated in FIG. 35B. Thus, the first net structure 500 a andthe second net structure 500 b are able to move in relation to eachother while the chain 810 maintains connection.

FIGS. 36A-36B illustrate an embodiment of a mechanical elementcomprising a hooking joint 820. In this embodiment, as shown in FIG.36A, the hooking joint 820 comprises a first hook 822 having a curvedshape and a second hook 824 also having a curved shape. The first hook822 and the second hook 824 are mateable so that the curved shapes hooktogether, such as illustrated. In some embodiments, the hooking joint820 is printed so that the first hook 822 is attached to a first netstructure 500 a and the second hook 824 is attached to a second netstructure 500 b, as illustrated in FIG. 36B wherein a plurality ofhooking joints (i.e., a first hooking joint 820 a, a second hookingjoint 820 b, a third hooking joint 820 c, and a fourth hooking joint 820d) are shown. Thus, the first net structure 500 a and the second netstructure 500 b are able to move in relation to each other while thefirst hooking joint 820 a, the second hooking joint 820 b, the thirdhooking joint 820 c, and the fourth hooking joint 820 d maintainconnection.

FIGS. 37A-37C illustrate an embodiment of a mechanical elementcomprising a hook-and-loop joint 830 which functions in a manner similarto Velcro®. In this embodiment, the hook-and-loop joint 830 comprises ahook surface 832 having a plurality of small hooks and a loop surface834 having a plurality of small loops. The hook surface 832 is mateablewith the loop surface 834 wherein the small hooks engage the smallloops, as illustrated in FIG. 37A, holding the hook surface 832 and theloop surface 834 together. However, the hook surface 832 and the loopsurface 834 may be disengaged by pulling the hook surface 832 and theloop surface 834 away from each other, as illustrated in FIG. 37B. Insome embodiments, the hook-and-loop joint 830 is printed so that thehook surface 832 is attached to a first net structure 500 a and the loopsurface 834 is attached to a second net structure 500 b, as illustratedin FIG. 37C. Thus, the first net structure 500 a and the second netstructure 500 b are joined and held in relation to each other byinteraction of the hook surface 832 and the loop surface 834 yet can bedisengaged with sufficient pulling force.

FIGS. 38A-38C illustrate an embodiment of a mechanical elementcomprising a hinge 840. In some embodiments, such as illustrated in FIG.38A, the hinge 840 comprises a first bracket 842 having a first bracketprotrusion 844 with a first bracket opening 846 therethrough, and asecond bracket 848 having a second bracket protrusion 850 with a secondbracket opening 852 therethrough. The hinge 840 further comprises a rod854, which is sized and configured to extend through the first bracketopening 846 and the second bracket opening 852. FIG. 38B illustrates thefirst bracket 842 and the second bracket 848 so that the rod 854 extendsthrough the first bracket opening 846 and the second bracket opening 852so as to join the first bracket 842 and the second bracket 848 togetherwhile allowing the first bracket 842 and the second bracket 848 toswivel and rotate, moving toward or away from each other. In someembodiments, the hinge 840 is printed so that the first bracket 842 isattached to a first net structure 500 a and the second bracket 848 isattached to a second net structure 500 b, as illustrated in FIG. 38C.Thus, the first net structure 500 a and the second net structure 500 bare joined and held in relation to each other by the hinge 840, yet canbe swivel and tilt in relation to each other.

As mentioned previously, the mechanical elements provide a variety offunctions, such as joining portions of a net and/or various netstructures together to create a complex three-dimensional tissuestructure, particularly in a manner which benefits from relationalmovement in the development process. This may assist in wrapping,looping, twisting or other desired movement within a net during thedevelopment process. FIG. 39 illustrates an embodiment of a tissuestructure comprised of cells 506 captured in a net structure 500 whereinthe net structure 500 is looping due to the presence of mechanicalelements. Similarly, FIG. 40 illustrates an embodiment of a tissuestructure comprised of cells 506 captured in net structure 500 whereinthe net structure 500 is twisting due to the presence of mechanicalelements.

In other embodiments, nets are linked or joined together by cells 506held in close proximity.

FIGS. 41A-41B illustrate an embodiment designed to induce cell-cellinteractions between two separate cell groups located in two separatenet structures. FIG. 41A illustrates a first net structure 500 a havinga first edge 900 and a second net structure 500 b having a second edge902 wherein the first edge 900 and the second edge 902 are in closeproximity. The first net structure 500 a and the second net structure500 b are printed having a first low density region 562 a bordering thefirst edge 900 and a second low density region 562 b bordering thesecond edge 902. Furthermore, the first net structure 500 a and thesecond net structure 500 b are printed having a first high densityregion 560 a bordering the first low density region 562 a and a secondlow density region 562 b bordering the second low density region 562 b.The first low density region 562 a and the second low density region 562b are sized to trap particular cells 506. The first high density region560 a and the second low density region 562 b, which are adjacent tofirst low density region 562 a and the second low density region 562 b,are sized to exclude cells 506. Thus, the cells 506 are held along thefirst edge 900 and the second edge 902 and favor cell-cell interactionswith each other, as illustrated in FIG. 41B. This binds the first edge900 and the second edge 902 together, linking or joining the first netstructure 500 a and the second net structure 500 b.

In other embodiments, variable density nets can be used to generate cellstrands, such as illustrated in FIG. 42A-42B. For example, as shown inFIG. 42A, in some embodiments, a net structure 500 is printed having alongitudinal region wherein the first apertures 504 are sized to trapparticular cells 506 and the surrounding second apertures 504′ are sizedto exclude cells 506. In such embodiments, the cells 506 are held inclose proximity within the longitudinal region and favor cell-cellinteractions with each other, creating a longitudinal strand of cells,as shown in FIG. 42B.

It may be appreciated that in some embodiments, nets structures includeelements that promote self-assembly, or structural elements that allowfor compression without primary structure deformation or other forceabsorbing, stretching elements to either restrict, facilitate, or allowmovement of cell sheets, strands, networks, groups, or individual cellsthrough structures that allow for cell “squeezing” and cytosolic flow.Likewise, some nets allow the formation or differentiation of cells inresponse to pressure, tension, progressive or pulsatile local shifts inmechanical forces of pressure, stretch, or tissue tension. In someinstances, movement, environmental responsiveness, and cell-cell contactwithin developing tissues is critical for functional organ, tissue, andcell development. Non-limiting examples of these include: individualcell-cell interactions that may or may not be part of a larger network.Movement of cells within coordinated networks that may include two orthree dimensional cell-sheet flow, folding, wrapping, deformation, ortwisting, or formation of strands, multi-layered spheroid formation orlinking necessary for functional tissue development and morphogenesis.

Beads containing bound or secreted signaling molecules, receptors,and/or stimulatory or blocking antibodies may be printed for promotingdirectional or localized self-assembly. Non-limiting examples of suchsignaling molecules include VEGF, to promote vascular outgrowth andbranching; VEGF-C, to promote lymphatic vasculature outgrowth anddevelopment; GDNF, to promote nerve development or, in kidney, uretericbud branching; or SHH, to promote tissue-dependent developmentalpatterning.

Such signaling molecule beads may also be used for directing axonpathfinding. In normal nerve development, the growth cone of adeveloping axon responds to attractive cues, which promote axonextension via assembly of cytoskeletal actin comprising the axon, andrepulsive cues, which prevent axon extension by inhibiting actinassembly and/or promoting actin disassembly. Both attractive andrepulsive cues are essential to proper pathfinding. Attractive cuesinclude EphrinB and netrins; repulsive cues include EphrinA,semaphorins, and Slit. Printing attractive and repulsive cues withinsignaling beads at desired locations provides a mechanism to promote andcontrol axonal growth from printed neural progenitor cells.

Print Media and Printing Conditions

As mentioned previously, the media chamber 122 contains media comprisedof cells, polymerizable material and culture medium. The polymerizablematerial comprises polymerizable monomeric units that are biologicallycompatible, dissolvable, and biologically inert. The monomeric unitspolymerize, cross-link or react in response to the multi-photon laserexcitation 120 to create cell containing structures, such as cellmatrices and basement membrane structures, specific to the tissue to begenerated. The media chamber may contain media comprising glutathione ora functional variant (or derivative) thereof.

In some embodiments, the media comprises a solution e.g. from at leastabout 0.2 mPa·s to about 10 Pa·s in viscosity, containing eitherphoto-activator or photo-activator-free polymerizable units. Thesolution may be doped with additional chemical and/or biologicalcomponents, with or without expressed chemical or biological activity,to alter the solution behavior such that it is non-Newtonian. Suchbehavior may be particularly useful in the instances of shear thinningproperties, wherein media becomes less viscous upon experiencing shearforce, or thixotropic media, wherein media becomes less viscous withvibration or shaking; such media may exhibit improved, better controlleddraining during media replacement. Non-limiting examples of suchcomponents that may be added to the cell-containing printing mediainclude extracellular matrix protein mixtures containing various amountsof hyaluronic acid, heparin sulfate, collagen types I through X,elastin, and fibrinogen. Additional organic or non-organic elements maybe introduced to the cell-containing print media to induce an increasedrate of avalanche ionization. Non-limiting examples of these particlesinclude non-toxic nanoparticles, moderate increases in elementalsubstances with a high number of freely available electrons such asselenium or lithium.

In some cases, specific conditions may be used during the printingprocess to facilitate the building of multiple cell layers usingmulti-photon printing of net structure 500 and components therein. Suchconditions provide for reduced cellular respiration, cell rounding,minimization of migration, and minimization of cellular damage, to namea few. In some cases, rounding of cells and reduction in adhesion may bedesired for trapping of cells in nets and efficient removal of cells nottrapped in the nets. This can be achieved by maintaining the temperatureof the printing media that contains polymerizable units or media thatdoes not contain additional cells or polymerizable monomers in a rangee.g. from about at least 1° C. to about 36° C. This temperature rangemay suppress cellular respiration, may encourage cell “rounding”, mayreduce laser induced temperature effects, and may minimize cellularmigration. The temperature may be controlled by either active or passivecooling mechanisms. Specifically, media or printing media used to createbiogels may be cooled by having a heat exchange platform for cellprinting or by printing in cooled ambient temperatures such as acold-room.

In general, infrared photons used for multiphoton printing may bediffuse and/or may be comprised of short, condensed, temporally distinctphoton packets. However, near the focal point, these photon packets maybecome increasingly condensed, resulting in a local increase in theconcentration of infrared (IR) radiation. Thus, the printing process canimpart heat to the surrounding media outside of the focal plane, anissue that increases in direct correlation to laser power increases.Therefore, heat as a function of infrared radiation, related eitherdirectly to the multi-photon wavelength itself or as part of thenon-radiative decay (energy loss of an excited electron prior to photonemission) can impart significant heat that can damage to cells. Coldprint-media may reduce this potential heat toxicity.

In some instances, highly localized increases in heat due to the energyassociated with high-intensity photon absorption near the focal pointmay lead to undesired polymerization or oxidation of some materials.Cooled media may assist in diffusing general infrared, focal point, andnear-focal point heat generation, thereby reducing potential heattoxicity to the living cells. In addition to reducing heat toxicity frominfrared radiation, cooled media may improve the structural rigidity ofmany polymerized materials and may increase the viscosity of printmedia, such that cells remain uniformly distributed. This increase instructural stiffness at cool temperatures and reduction of flexibilitymay allow improved rates of cell-containing media exchange foradditional rounds of printing without damage to the deposited structure.

In some instances, highly localized increases in heat due to the energyassociated with high-intensity photon absorption near the focal pointmay lead to polymerization or oxidation of some materials. In someformulations of monomer and cell containing print materials, highlylocalized increases in heat may be desirable, as many biocompatiblemonomers can be polymerized into strands to create cell nets. Thisprocess can be tuned by using radiative heat emission at differentwavelengths and polymerization of monomers may be specific only tothermal radiation. In some formulations this may be achieved by usingheat polymerized compounds that are otherwise non-responsive to photonabsorption, light-induced printing, and/or photopolymerization.

Removal of cations, such as calcium and/or magnesium, by addition ofchelating agents can reduce protein-protein interactions between cellsand between cells and the extracellular matrix, reducing migration,promoting cell rounding, and temporarily slowing or speeding up theprogression of cell differentiation. Therefore, in some cases, the printbiogel and media may be kept within a range e.g. from at least about 0to about 1.8 nanomolar (nM) calcium concentrations. Either naturally lowcalcium concentration media may be used or the addition of calciumchelating agents may be incorporated in the cell containing print media.In some cases, cation chelating agents may be added to reduce molarconcentrations of cations including but not limited to calcium,magnesium, and, or sodium. Non-limiting examples of chelating agentsinclude ethylenediaminetetraacetic acid (EDTA), ethyleneglycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), and1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA). Insome cases, naturally occurring small molecules and chemicals releasedby cells are added to specific formulations of print media to facilitatereduced cellular respiration, facilitate cell respiration recovery, orquench radicals generated by the printing process. Non-limiting examplesinclude hydrogen sulfide (H₂S), sodium hydro sulfide (NaHS), nitrousoxide, glutathione, phosphate, β-glycerophosphate, sodium pyruvate,L-glutamine, carbon-based sugars, micronutrients, mixed human serumproteins and growth factors, metabolic effectors (insulin), cytokines,chemokines, and compounds that interact with internal cell pathways suchas the Rho/Rac pathway, PI3-kinase pathways, or ubiquitinase inhibitors.Once the printing process is complete or partially complete, the mediasurrounding the newly printed structure may be returned to physiologicconditions, to allow for cells to return to normal homeostatic functionand active motility.

In some cases, glutathione or a functional variant (or derivative)thereof may be added to a formulation of print media (i.e., to themedium). Glutathione (GSH) is an important antioxidant in livingorganisms; it is a cellular-health promoting free-radical scavenger.Glutathione may prevent cellular damage caused by reactive oxygenspecies, such as but not limited to free radicals, peroxides, lipidperoxides, and/or heavy metals. Glutathione or a functional variant (orderivative) thereof may be used in a manufacturing process and/or in aprinting process. Glutathione is a free-radical inhibitor that may beused in a manufacturing process and/or a printing process which includescells. Glutathione or a functional variant (or derivative) may be usedin a manufacturing process and/or a printing process that uses cells. Insome cases, glutathione or a functional variant (or derivative) thereofmay quench radicals generated by the 3D holographic printing process.Glutathione or a functional variant (or derivative) thereof may suppressany additional polymerization outside of a desired print area byquenching a radical reaction. The methods and systems provided hereinmay use glutathione or a functional variant (or derivative) thereof forcontrolling a polymerization reaction during the 3D holographic printingprocess in order to achieve the printing of ultra-fine architecturenecessary for tissue engineering. Functional variants and/or derivativesof glutathione may include, but are not limited to sodium pyruvate andL-glutamine.

The medium may further comprise glutathione or a functional variant (orderivative) thereof. The medium may comprise at least about 0.1millimolar (mM) to about 50 mM or more of glutathione or a functionalvariant (or derivative) thereof. The medium may comprise at least about0.01 millimolar (mM) to about 50 mM or more of glutathione or afunctional variant (or derivative) thereof. The medium may comprise atleast about 0.05 millimolar (mM) to about 50 mM or more of glutathioneor a functional variant (or derivative) thereof. The medium may compriseat least about 0.5 millimolar (mM) to about 50 mM or more of glutathioneor a functional variant (or derivative) thereof. The medium may compriseat least about 1 millimolar (mM) to about 50 mM or more of glutathioneor a functional variant (or derivative) thereof. The medium may compriseat least about 5 millimolar (mM) to about 50 mM or more of glutathioneor a functional variant (or derivative) thereof. The medium may compriseat least about 10 millimolar (mM) to about 50 mM or more of glutathioneor a functional variant (or derivative) thereof. The medium may compriseat least about 20 millimolar (mM) to about 50 mM or more of glutathioneor a functional variant (or derivative) thereof. The medium may compriseat least about 30 millimolar (mM) to about 50 mM or more of glutathioneor a functional variant (or derivative) thereof. The medium may compriseat least about 40 millimolar (mM) to about 50 mM or more of glutathioneor a functional variant (or derivative) thereof.

The medium may comprise at least about 0.01 mM of glutathione or afunctional variant (or derivative) thereof. The medium may comprise atleast about 0.02 mM of glutathione or a functional variant (orderivative) thereof. The medium may comprise at least about 0.03 mMglutathione or a functional variant (or derivative) thereof. The mediummay comprise at least about 0.04 mM glutathione or a functional variant(or derivative) thereof. The medium may comprise at least about 0.05 mMglutathione or a functional variant (or derivative) thereof. The mediummay comprise at least about 0.06 mM glutathione or a functional variant(or derivative) thereof. The medium may comprise at least about 0.07 mMglutathione or a functional variant (or derivative) thereof. The mediummay comprise at least about 0.08 mM glutathione or a functional variant(or derivative) thereof. The medium may comprise at least about 0.09 mMglutathione or a functional variant (or derivative) thereof. The mediummay comprise at least about 0.1 mM glutathione or a functional variant(or derivative) thereof. The medium may comprise at least about 0.2 mMglutathione or a functional variant (or derivative) thereof. The mediummay comprise at least about 0.3 mM glutathione or a functional variant(or derivative) thereof. The medium may comprise at least about 0.4 mMglutathione or a functional variant (or derivative) thereof. The mediummay comprise at least about 0.5 mM glutathione or a functional variant(or derivative) thereof. The medium may comprise at least about 0.6 mMglutathione or a functional variant (or derivative) thereof. The mediummay comprise at least about 0.7 mM glutathione or a functional variant(or derivative) thereof. The medium may comprise at least about 0.8 mMglutathione or a functional variant (or derivative) thereof. The mediummay comprise at least about 0.9 mM glutathione or a functional variant(or derivative) thereof. The medium may comprise at least about 1 mMglutathione or a functional variant (or derivative) thereof. The mediummay comprise at least about 2 mM glutathione or a functional variant (orderivative) thereof. The medium may comprise at least about 3 mMglutathione or a functional variant (or derivative) thereof. The mediummay comprise at least about 4 mM glutathione or a functional variant (orderivative) thereof. The medium may comprise at least about 5 mMglutathione or a functional variant (or derivative) thereof. The mediummay comprise at least about 6 mM glutathione or a functional variant (orderivative) thereof. The medium may comprise at least about 7 mMglutathione or a functional variant (or derivative) thereof. The mediummay comprise at least about 8 mM glutathione or a functional variant (orderivative) thereof. The medium may comprise at least about 9 mMglutathione or a functional variant (or derivative) thereof. The mediummay comprise at least about 10 mM glutathione or a functional variant(or derivative) thereof. The medium may comprise at least about 15 mMglutathione or a functional variant (or derivative) thereof. The mediummay comprise at least about 20 mM glutathione or a functional variant(or derivative) thereof. The medium may comprise at least about 25 mMglutathione or a functional variant (or derivative) thereof. The mediummay comprise at least about 30 mM glutathione or a functional variant(or derivative) thereof. The medium may comprise at least about 35 mMglutathione or a functional variant (or derivative) thereof. The mediummay comprise at least about 40 mM glutathione or a functional variant(or derivative) thereof. The medium may comprise at least about 45 mMglutathione or a functional variant (or derivative) thereof. The mediummay comprise about 50 mM glutathione or a functional variant (orderivative) thereof or more. The medium may comprise at least about 75mM glutathione or a functional variant (or derivative) thereof. Themedium may comprise at least about 100 mM glutathione or a functionalvariant (or derivative) thereof.

Together, low calcium and cold temperatures may have important effectson cell behavior, metabolic processes, and physiologic responses totheir environment that may be critical for multi-layered tissueprinting. These may include, but are not limited to: i) cells maintainedin low calcium (Ca²⁺) concentrations and cold media take on a roundshape and withdraw protrusions; ii) cellular low calcium (Ca²⁺) and coldmedia conditions functionally alter integrins, mucins, (and otherproteins) are functionally altered by. Low Ca²⁺ concentrations may alterphysical protein conformations, such that cell adhesion is significantlyreduced if not completely absent. In addition, cold temperatures mayreduce veracity of protein-protein interactions. iii) Low Ca²⁺, coldmedia may halt signaling associated with external cell-cell interactionsand intrinsic cell signaling associated with environmental responses andgenetic changes; and iv) reduced propensity for cell-cell interactionsmay allow for high-density single-cell suspensions with low or no cellaggregate formation. Reduction or minimization of cell aggregateformation may be critical for even cell distribution and placementwithin confined structures.

Together, these conditions may cause important physiologic changes ofrounding and reduced matrix-cell and cell-cell interactions, reducedcellular respiration, and biochemical support of cellular respirationfunctions through CO₂ buffering. CO₂ buffering can be achieved by addingvarious small molecules or agents to the cell-containing print media.

Additionally, changes in pH can significantly alter viscosity, cellsurvival, or print media properties. Therefore, changes to orstabilization of the pH of the cell-containing media, biogel, or printmaterial may be effected by addition of various pH buffers. In someinstances, print media pH may be critical for health and function ofcells during the print process and during the recovery period.Therefore, in some cases, buffers that assist in controlling the pH ofthe cell print media may be included in the media. Such pH buffers maybe added to reduce pH changes or fluctuations related to the printingprocess, cellular respiration, or other components that may be added toprint media. Non-limiting examples of cell compatible and printcompatible pH buffers may include: 2-(N-morpholino)ethanesulfonic acid(MES), bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane (BIS-TRIS),2-[(2-amino-2-oxoethyl)-(carboxymethyl)amino]acetic acid (ADA),2-(carbamoylmethylamino)ethanesulfonic acid (ACES),1,3-bis(tris(hydroxymethyl)methylamino)propane (BIS-TRIS PROPANE),piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES),N-(2-Acetamido)-2-aminoethanesulfonic acid (ACES),2-hydroxy-3-morpholin-4-ylpropane-1-sulfonic acid (MOPSO), cholaminechloride, 3-(N-morpholino)propanesulfonic acid (MOPS),N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES),2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid(TES), 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES),3-(N,N-Bis[2-hydroxyethyl]amino)-2-hydroxypropanesulfonic acid (DIPSO),4-(N-Morpholino)butanesulfonic acid (MOBS),2-Hydroxy-3-[tris(hydroxymethyl)methylamino]-1-propanesulfonic acid(TAPSO), acetamidoglycine, Tris-acetate-ethylenediaminetetraacetic acid(TAE), piperazine-1,4-bis(2-hydroxypropanesulfonic acid) dihydrate(POPSO), 4-(2-hydroxyethyl)piperazine-1-(2-hydroxypropanesulfonic acid)(HEPPSO), 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid (EPS),4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid (HEPPS), tricine,2-amino-2-(hydroxymethyl)-1,3-propanediol (TRIZMA), glycinamide,glycyl-glycine, N-(2-hydroxyethyl)piperazine-N′-(4-butanesulfonic acid)(HEPBS), bicine,3-{[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino}propane-1-sulfonicacid (TAPS), 2-amino-2-methyl-1-propanol buffer (AMPB),2-(cyclohexylamino)ethanesulfonic acid (CHES),N-(1,1-Dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid(AMPSO), 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO),3-(cyclohexylamino)-1-propanesulfonic acid (CAPS),4-(cyclohexylamino)-1-butanesulfonic acid (CABS).

Additional methods are provided to enhance the printing of polymersprocess, through photon-based and thermally-induced polymerizationprocess around cells as related to three-dimensional projection ofmulti-photon excitation. First, biologically compatible or biologicallyinert electron donors may enhance electron cascade phenomenon, which mayincrease the rate of multiphoton based polymerization. In some cases,this phenomenon may be utilized with the use of biogels or printingmaterials containing cells having specific properties, such as havingelectron shells with close energy states for ease of transition betweenground and excited states. Enhancing the effect or likelihood ofelectron cascade initiation may be achieved by adding additionalelements to the biogel to serve as ready electron donors into thesystem.

In some cases, the speed of bioprinting tissue may be enhanced by dopingof cell-compatible electron donors as activators for the purpose ofgenerating electron cascade events, tuning the dynamic range ofphotopolymerization, or selecting of multi-photon wavelengths that doso. These electron donors may include dyes, nanoparticles, orbiologically active electron donors, including but not limited to ionssuch as lithium, selenium, iodine, or larger organic molecules such asnicotinic acid and riboflavins. Doping of biogels may also expand therange of sensitivity for photon-based polymerization such thatpolymerization may occur as a result of energy transfer from theparticle, molecule, or compound used as a doping agent to inducepolymerization. Photon cascade may also be used in the case oftwo-photon polymerization, wherein a doping particle may be selected forits ability to release light of different wavelengths based on randomand alternative paths towards ground state.

Furthermore, in some cases, tuning of the dynamic range forpolymerization may allow for additional structural properties to beadded to cell nets, including relatively increased or decreased regionsof polymer density just by changing the duration of excitation orintensity of excitation both of which increase the voxel size. Thisincrease of density or thickness within the same print pass may beachieved by projecting or flickering off and on only certain portions orcomponents of three dimensional images such that specifically selectedspots or regions in the structure experience extended laser exposuretimes, allowing for introduction of varied structural elements.

Together, these features may allow for extended print-times whileprinting larger or more structures, longer-lasting and more uniform celldispersion and distribution in suspension, with minimal damage to cells.Additionally, these conditions may facilitate more complete removal ofcells not immobilized in cell nets during the multi-layered printingprocesses. Together, these print media conditions may allow for morecontrolled placement of cells and increased cell survival and facilitateremoval during extended time periods required for multiple rounds ofcell containing structure deposition.

Computer Control Systems

The present disclosure provides computer control systems that areprogrammed to implement methods of the disclosure. FIG. 43 shows acomputer system 1101 that is programmed or otherwise configured toreceive a computer model of the 3D biological material in computermemory; generate a point-cloud representation or lines-basedrepresentation of the computer model of the 3D biological material incomputer memory; and direct the at least one energy source to direct theenergy beam to the medium in the media chamber along at least one energybeam path in accordance with the computer model of the 3D biologicalmaterial, and to subject at least a portion of the polymer precursors toform at least a portion of the 3D biological material. The computersystem 1101 can regulate various aspects of computer model generationand design, image generation, holographic projection, and lightmodulation of the present disclosure, such as, for example, receiving orgenerating a computer-aided-design (CAD) model of a desiredthree-dimensional (3D) biological material structure to be printed. Thecomputer system 1101 can convert the CAD model or any other type ofcomputer model such as a point-cloud model or a lines-based model intoan image of the desired three-dimensional (3D) biological materialstructure to be printed. The computer system 1101 can project the imagethe desired three-dimensional (3D) biological material structureholographically. The computer system 1101 can modulate a light source,an energy source, or an energy beam such that a light path or an energybeam path is created by the computer system 1101. The computer system1101 can direct the light source, the energy source, or the energy beamalong the light path or the energy beam path. The computer system 1101can be an electronic device of a user or a computer system that isremotely located with respect to the electronic device. The electronicdevice can be a mobile electronic device.

The computer system 1101 includes a central processing unit (CPU, also“processor” and “computer processor” herein) 1105, which can be a singlecore or multi core processor, or a plurality of processors for parallelprocessing. The computer system 1101 also includes memory or memorylocation 1110 (e.g., random-access memory, read-only memory, flashmemory), electronic storage unit 1115 (e.g., hard disk), communicationinterface 1120 (e.g., network adapter) for communicating with one ormore other systems, and peripheral devices 1125, such as cache, othermemory, data storage and/or electronic display adapters. The memory1110, storage unit 1115, interface 1120 and peripheral devices 1125 arein communication with the CPU 1105 through a communication bus (solidlines), such as a motherboard. The storage unit 1115 can be a datastorage unit (or data repository) for storing data. The computer system1101 can be operatively coupled to a computer network (“network”) 1130with the aid of the communication interface 1120. The network 1130 canbe the Internet, an internet and/or extranet, or an intranet and/orextranet that is in communication with the Internet. The network 1130 insome cases is a telecommunication and/or data network. The network 1130can include one or more computer servers, which can enable distributedcomputing, such as cloud computing. The network 1130, in some cases withthe aid of the computer system 1101, can implement a peer-to-peernetwork, which may enable devices coupled to the computer system 1101 tobehave as a client or a server.

The CPU 1105 can execute a sequence of machine-readable instructions,which can be embodied in a program or software. The instructions may bestored in a memory location, such as the memory 1110. The instructionscan be directed to the CPU 1105, which can subsequently program orotherwise configure the CPU 1105 to implement methods of the presentdisclosure. Examples of operations performed by the CPU 1105 can includefetch, decode, execute, and writeback.

The CPU 1105 can be part of a circuit, such as an integrated circuit.One or more other components of the system 1101 can be included in thecircuit. In some cases, the circuit is an application specificintegrated circuit (ASIC).

The storage unit 1115 can store files, such as drivers, libraries andsaved programs. The storage unit 1115 can store user data, e.g., userpreferences and user programs. The computer system 1101 in some casescan include one or more additional data storage units that are externalto the computer system 1101, such as located on a remote server that isin communication with the computer system 1101 through an intranet orthe Internet.

The computer system 1101 can communicate with one or more remotecomputer systems through the network 1130. For instance, the computersystem 1101 can communicate with a remote computer system of a user.Examples of remote computer systems include personal computers (e.g.,portable PC), slate or tablet PC's (e.g., Apple® iPad, Samsung® GalaxyTab), telephones, Smart phones (e.g., Apple® iPhone, Android-enableddevice, Blackberry®), cloud based computing services (e.g. Amazon WebServices), or personal digital assistants. The user can access thecomputer system 1101 via the network 1130.

Methods as described herein can be implemented by way of machine (e.g.,computer processor) executable code stored on an electronic storagelocation of the computer system 1101, such as, for example, on thememory 1110 or electronic storage unit 1115. The machine executable ormachine readable code can be provided in the form of software. Duringuse, the code can be executed by the processor 1105. In some cases, thecode can be retrieved from the storage unit 1115 and stored on thememory 1110 for ready access by the processor 1105. In some situations,the electronic storage unit 1115 can be precluded, andmachine-executable instructions are stored on memory 1110.

The code can be pre-compiled and configured for use with a machinehaving a processor adapted to execute the code, or can be compiledduring runtime. The code can be supplied in a programming language thatcan be selected to enable the code to execute in a pre-compiled oras-compiled fashion.

Aspects of the systems and methods provided herein, such as the computersystem 1101, can be embodied in programming. Various aspects of thetechnology may be thought of as “products” or “articles of manufacture”typically in the form of machine (or processor) executable code and/orassociated data that is carried on or embodied in a type of machinereadable medium. Machine-executable code can be stored on an electronicstorage unit, such as memory (e.g., read-only memory, random-accessmemory, flash memory) or a hard disk. “Storage” type media can includeany or all of the tangible memory of the computers, processors or thelike, or associated modules thereof, such as various semiconductormemories, tape drives, disk drives and the like, which may providenon-transitory storage at any time for the software programming. All orportions of the software may at times be communicated through theInternet or various other telecommunication networks. Suchcommunications, for example, may enable loading of the software from onecomputer or processor into another, for example, from a managementserver or host computer into the computer platform of an applicationserver. Thus, another type of media that may bear the software elementsincludes optical, electrical and electromagnetic waves, such as usedacross physical interfaces between local devices, through wired andoptical landline networks and over various air-links. The physicalelements that carry such waves, such as wired or wireless links, opticallinks or the like, also may be considered as media bearing the software.As used herein, unless restricted to non-transitory, tangible “storage”media, terms such as computer or machine “readable medium” refer to anymedium that participates in providing instructions to a processor forexecution.

Hence, a machine readable medium, such as computer-executable code, maytake many forms, including but not limited to, a tangible storagemedium, a carrier wave medium or physical transmission medium.Non-volatile storage media include, for example, optical or magneticdisks, such as any of the storage devices in any computer(s) or thelike, such as may be used to implement the databases, etc. shown in thedrawings. Volatile storage media include dynamic memory, such as mainmemory of such a computer platform. Tangible transmission media includecoaxial cables; copper wire and fiber optics, including the wires thatcomprise a bus within a computer system. Carrier-wave transmission mediamay take the form of electric or electromagnetic signals, or acoustic orlight waves such as those generated during radio frequency (RF) andinfrared (IR) data communications. Common forms of computer-readablemedia therefore include for example: a floppy disk, a flexible disk,hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD orDVD-ROM, any other optical medium, punch cards paper tape, any otherphysical storage medium with patterns of holes, a RAM, a ROM, a PROM andEPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wavetransporting data or instructions, cables or links transporting such acarrier wave, or any other medium from which a computer may readprogramming code and/or data. Many of these forms of computer readablemedia may be involved in carrying one or more sequences of one or moreinstructions to a processor for execution.

The computer system 1101 can include or be in communication with anelectronic display 1135 that comprises a user interface (UI) 1140 forproviding, for example, status of the printing process (e.g. displayingan illustration of the 3D biological material representing the 3D tissueportions printed prior to completion of the process), manual controls ofthe energy beams (e.g. emergency stop buttons controlling the on/offstates of the energy beam), and display indicators designed to e.g.display remote oxygen, carbon dioxide, humidity, and temperaturemeasurements within the media chamber. Examples of UI's include, withoutlimitation, a graphical user interface (GUI) and web-based userinterface.

EXAMPLES Example 1—Holographic Printing of a Biologically FunctionalAortic Valve Using a Method and System Described Herein

In an example, a patient presents with symptoms such as shortness ofbreath, chest pain, and a heart murmur. A physician diagnoses thepatient with aortic valve stenosis and recommends aortic valvereplacement surgery. The patient undergoes a computer tomography (CT)scan of the aortic valve. The CT scan of the aortic valve is thenconverted into a computer-aided-design (CAD) model, which is received bythe computer processor of the system disclosed herein. The computerprocessor generates a point-cloud representation of the aortic valve CADmodel in computer memory. The computer processor further converts thepoint-cloud representation of the aortic valve into an image such as athree-dimensional image. The system further deconstructs andreconstructs the three-dimensional image and projects it in aholographic manner in a media chamber. The media chamber comprises cellssuch as fibroblasts, cartilage supporting chondrocytes, and partiallydifferentiated mesenchymal stem cells; cell culture medium such asCardiomyocyte Maintenance Medium; a polymerizable material (e.g., 2mg/mL collagen methacrylate and 50% w/v polyethylene glycol diacrylate(PEGDA)); and a photoinitiator (e.g., Eosin Y Next, the system directsat least one energy beam to the media chamber along at least one energybeam path in accordance with the point-cloud representation of theaortic valve of the patient to subject the polymerizable material toform a 3D biologically functional aortic valve. The 3D-printedbiologically functional aortic valve is then grown in tissue culturemedia conditions, assessed for functional and structural properties, andultimately used to replace the diseased aortic valve of the patientduring the aortic valve replacement surgery.

Example 2—Holographic Printing of a Vascularized, Three-Dimensional SkinTransplant

In another example, a physician treats a patient for a severe skindisorder or burn and a replacement tissue is needed. The physician takesa skin biopsy between about 3 mm and 10 cm, depending upon initial cellnumber requirement, from a portion of healthy skin that is either fromthe patient or a genetic match of the patient. The physician sends theskin biopsy to Prellis Biologics. Prellis Biologics dissociates theskin; i.e. Prellis Biologics grows, and expands several of the distinctcell types from the skin such as, but not limited to keratinocytes,fibroblasts, epithelial cells, and stem cells of various differentiationstates until sufficient numbers of cells are obtained to print newvascularized skin. A model of vascularized skin and the order of layerprinting are loaded into a computer system that controls the opticalelements that guide the laser or energy beam to the media chamber where3D printing of new skin occurs. The order of cells to be printed isdetermined, for example, vascular cells are printed first. Small bloodvessels are printed using the methods and systems described herein. Thecell-containing medium comprises endothelial cells and a mixture of 1mg/mL collagen methacrylate and 50% w/v polyethylene glycol diacrylate(PEGDA). The cell-containing medium may be a bio-ink. Once vasculatureis printed, the printed structure is removed from the cell-containingmedium and maintained at physiologic conditions until there is a stablevascular system. Next, the stability of the printed vascular system isverified by fluid flow tests, and the remaining cell types that arepresent in skin layers such as, but not limited to keratinocytes,epithelial cells, stem cells, and/or fibroblasts are printed around theexisting printed vasculature, to form dermal and epidermal layers. Theremaining cell types are also printed using the methods and systemsdescribed herein. Intradermal structures, such as, but not limited tohair follicles and sebaceous glands, are printed around the previouslyprinted three-dimensional structure using a cell-containing mediumcomprising epithelial stem cells; thus, a printed three-dimensional skinstructure is formed. The printed three-dimensional skin structure isthen returned to physiologic conditions provided by the cell culturesystems. The three-dimensional printed skin structure is supplied withits own perfusion system via pumping of nutrient rich, oxygenated mediaand/or blood substitute through the plurality of lumens of the vascularsystem. Differentiation and growth of the three-dimensional printed skinstructure is monitored by an occasional biopsy and when a sufficientdevelopmental state is reached, the three-dimensional printed skinstructure is returned to the physician for transplantation. Thevascularized, three-dimensional, printed skin transplant describedherein has many benefits over other solutions, including, but notlimited to the fact that the tissue is living and surgical anastomosis,or connection of blood vessels with the patient's own circulatorysystem, allows for functional incorporation of the graft.

Example 3—Holographic Printing of a Three-Dimensional, FunctionalPrinted Kidneys

In another example, a patient presenting with kidney failure isundergoing dialysis three times a week to remove waste and extra fluidfrom blood. A physician takes a kidney biopsy from the patient or amatched healthy donor kidney and provides the kidney biopsy to PrellisBiologics. Prellis Biologics cultures cells extracted from the kidneybiopsy and expands adult kidney progenitor cell populations, including,but not limited to mesenchymal stem cells and dedifferentiated tubularepithelial cells, in vitro. A renal capillary system is printed from CADmodels of an adult kidney vasculature system, using laser-initiatedpolymerization of a cell-containing medium comprising endothelial cells,and mixture of 1 mg/mL collagen methacrylate and 50% w/v polyethyleneglycol diacrylate (PEGDA). Printed vasculature is maintained underphysiological conditions, using endothelial cell culture media, untilfunctional vasculature is demonstrated. Once functional vasculature isdemonstrated, tubule structures of the nephron are printed within andaround the vasculature system, using a cell-comprising medium includingmesenchymal stem cells, tubular epithelial cells, and photosensitiveextracellular matrix (ECM) components. ECM components are printed into3D convoluted tubule structures with a plurality of perfusable, openlumens. Mesenchymal stem cells and tubular epithelial cells form aconfluent epithelial monolayer around the ECM scaffolding. Controlledperfusion of morphogens and growth factors, combined with the unique 3Dgeometry of the printed tubules, directs differentiation into mature,polarized kidney epithelial cells forming each of the components of afunctional nephron. Functional printed kidneys comprise at least 200,000nephron units. Functionality and tissue viability is tested prior totransplantation into the patient. The three-dimensional, functionalprinted kidneys are returned to the physician for transplantation intothe patient.

Example 4—Holographic Printing of a Cellularized, Three-Dimensional(3D), Impermeable Microvasculature Structure

In another example, the 3D printing methods and systems provided hereinwere used to print a cellularized, 3D, impermeable microvasculaturestructure, as shown in FIGS. 48A-48E. FIG. 48A shows a top-down view ofthe 3D microvasculature structure in one of the initial steps ofprinting. A multi-photon energy beam 120 was used to project (in atop-down manner) a hologram of the 3D microvasculature structure into amedium. The medium contained cell culture medium, collagen methacrylate,PEGDA, Eosin Y, and cells 506. The cells 506 included endothelial cells.The inner tube 104 of the 3D microvasculature structure had a diameterof about 10 microns (μm) and was completed in the early steps of the 3Dprinting process, as shown in FIG. 48A. FIG. 48B shows a top-down viewof the outer tube 102 of the 3D microvasculature structure as it beganto form later in the process. The outer tube 102 had a diameter of about50 μm. The completed 3D blood vessel structure was polymerized in situ,forming the inner tube 104 inside the outer tube 102 while trappingcells 506 around its structure, as shown in FIG. 48C. The final tubelength that was holographically printed ranged from about 250 to 300 μmlong. FIG. 48D is a fluorescent image of three microvasculaturestructures comprising an inner tube 104 and an outer tube 102. The three3D printed microvasculature structures showed fluorescently-labeledcells 506 trapped within the microvasculature structures. FIG. 48E showsa bright field image of three 3D printed microvasculature structurescontaining cells 506. The three 3D printed microvasculature structureswere placed under physiologic cell-culture conditions and imaged inbright field on day 5 after holographic printing. The 3Dmicrovasculature structures contained dye (darker areas shown in FIG.48E) after 5 days in culture, indicating the 3D microvasculaturestructures were impermeable to small molecules and whole cells wereretained inside the printed microvasculature structures.

Example 5—Generation of a Cell-Containing Structure Using HolographicPrinting

In another example, the 3D printing methods and systems provided hereinwere used to print a cell-containing structure, as shown in FIGS.49A-49H. FIG. 49A shows a computer generated three-dimensional (3D)image of a cell-containing structure. A computer processor was thenprogrammed to generate a point-cloud representation of the 3D image ofthe cell-containing structure shown in FIG. 49B. The computer processorwas further programmed by the algorithms provided herein to convert thepoint-cloud representation into the hologram shown in FIG. 49C. Thepoint-cloud representation and the hologram were used to generatecomputer instructions for printing the 3D cell-containing structure;these computer instructions were relayed to the computer printing systemshown in FIG. 49D. A laser beam was directed into a media chamber (notshown in FIG. 49) containing a cluster of living cells 506 suspended inliquid print media 126, which included at least one polymeric precursor,as shown in FIG. 49E. FIG. 49F shows the same cluster of living cells506 after three dimensional printing of the point-cloud representation.The printing field in this example was centered on the cell cluster bythe user. FIG. 49G shows a cut-away image showing cells 506 inside ofthe printed, 3D cell-containing structure. FIG. 49H shows arepresentative image of the complete print of the 3D cell-containingstructure. The entire cell-containing structure was printed in about 7seconds.

Example 6—Holographic Printing of a 3D “Stanford Bunny”

In another example, the 3D printing methods and systems provided hereinwere used to print a three-dimensional (3D) “Stanford Bunny” structure,as shown in FIGS. 50A-50C. The “Stanford Bunny” is a common computergraphics 3D test model. FIG. 50A shows a computer generatedthree-dimensional (3D) image of the “Stanford Bunny.” FIG. 50B shows atop-down view of the computer generated 3D image of the “StanfordBunny.” A computer processor was programmed to generate a point-cloudrepresentation of the 3D image of the “Stanford Bunny,” and thepoint-cloud representation was converted into a hologram. A laser beamwas directed into a media chamber containing liquid print mediaincluding at least one polymeric precursor (not shown in FIG. 50). FIG.50C shows a representative 3D print of the “Stanford Bunny” as imagedusing in bright-field microscopy. The entire 3D structure of the“Stanford Bunny” was printed in about 60 seconds.

Example 7—Demonstration of Holographic Printing as a TwoPhoton-Dependent Process

In another example, FIGS. 51A-51B show graphs of a two-photon laser beamexposure time (in milliseconds) vs. laser power (Watts) corresponding toholographic printing of two different formulations. Two-photonabsorption is a second-order process wherein two photons of identical ordifferent frequencies are absorbed in order to excite a molecule fromone state to a higher electronic state. FIGS. 51A-51B demonstrate theprocess of holographic printing as a two photon dependent process;wherein the two-photon laser exposure time to the print sample wascontrolled by a computer processor that dictated the rapid opening andclosing of the laser shutter to match the described time period and thethreshold for printing. Per the standard two-photon absorptive process,the exposure time necessary to print is proportional to the inherentprinting material properties divided by the power squared. FIG. 51Ashows the threshold for printing in Formulation A which comprised atleast about 30% PEG-DA, 0.5% Eosin Y, and 1 mg/mL collagen diacrylate.Extrapolation of the raw data points in a log scale fitted a lineardecay, as shown in the graph on the right in FIG. 51A. The linear decayof Formulation A, shown in the log scale graph, matched the linear decaymodel that is expected for a second order process. FIG. 51B shows thethreshold for printing in Formulation B which comprised at least about45% PEG-DA, 0.5% Eosin Y, and 1 mg/mL collagen diacrylate. Extrapolationof the raw data points of Formulation B in a log scale fitted a lineardecay, as shown in the graph on the right in FIG. 51B. The linear decayof Formulation B, shown in the log scale graph, corresponded to thelinear decay model that is expected for a second order process.

Example 8—Targeted Single Cell Encapsulation Using Holographic Printing

In another example, the 3D printing methods and systems provided hereinwere used to perform a targeted single cell encapsulation, as shown inFIGS. 52A-52C. FIG. 52A shows a plurality of encapsulated cells andnon-encapsulated cells suspended in print media comprising at least onepolymeric precursor. For example, a first encapsulated cell 142 a, asecond encapsulated cell 142 b, a third encapsulated cell 142 c, a firstnon-encapsulated cell 144 a, a second non-encapsulated cell 144 b, and athird non-encapsulated cell 144 c are shown in FIG. 52A. FIG. 52B showszoomed-in images of a first encapsulated cell 142 a, a secondencapsulated cell 142 b, and a third encapsulated cell 142 c. Thesecells were encapsulated by 3D polymeric spheres with a diameter of about25 microns (μm) that were printed holographically using the methods andsystems provided herein. FIG. 52C shows zoomed-in images of a firstnon-encapsulated cell 144 a, a second non-encapsulated cell 144 b, and athird non-encapsulated cell 144 c. The non-encapsulated cells were notsubjected to holographic printing of a 3D sphere around them. The 3Dhologram was projected onto an individual cell (e.g., onto the firstencapsulated cell 142 a) for at most about 50 milliseconds (ms) perencapsulation event.

Example 9—Expanded Laser Beam Projecting a Holographic Image

In another example, a representative image of the 3D printing system isshown in FIG. 53; in particular, the expanded laser beam projecting aholographic image is shown. FIG. 53 shows a laser beam having awavelength of 1035 nm (i.e., a wavelength in the far-red light spectrum)as it was projected as an expanded laser beam that was patterned in aholographic form onto the back aperture of the print head. In thisembodiment, the print head was a standard physiology grade microscopeobjective. The image shown in FIG. 53 was taken using long exposurewhile an infrared-detecting laser card was run through the light path toilluminate the light path in the visible range.

Example 10—Various Laser Printing Modes

In another example, FIGS. 54A-54D illustrate different laser printingmodes based on the optics of single photon and multiphoton printingprocesses and the expected structural outcomes. FIG. 54A illustrates asingle photon laser beam projection into a media chamber containing aphotosensitive print medium. The single photon laser beam projection isshown in FIG. 54A without masking or isolation of the intended plane offocus, which may be expected to leave a printed structure behind in theshape of the entire light cone. FIG. 54B illustrates a multi-photonabsorption process where the photon density is only high enough at thepoint of focus, leaving only a pin-point structure behind in a mediachamber containing a photosensitive print medium. FIG. 54C illustrates arepresentative graphic of wavefront shaping to produce a hologram inwhich the multiphoton absorption process occurs at multiple points offocus in the x, y, and z planes. In this embodiment, rapid switchingbetween 3D projected hologram portions of a complete structure may beused to build the complete structure. FIG. 54D illustrates a completeimage projection (i.e., a 3D hologram) in multiple planes allowing forthe holographic printing of a complex structure. The complex structureshown in FIG. 54D as an example is a microvasculature structure havingan inner tube and an outer tube.

Example 11—Holographic Printing of Spheres within a Previously Printed3D Microvasculature Structure

In another example, the 3D printing methods and systems provided hereinwere used to print spheres inside a previously printed 3Dmicrovasculature structure, as shown in FIGS. 55A-55F. FIG. 55Aillustrates a printed microvasculature structure comprising a hollowtube structure and corresponds to the image shown in FIG. 55B. FIG. 55Bshows an image of a printed microvasculature structure prior to theprinting of a sphere. As shown in FIGS. 55C-55D, a multi-photon energybeam 120 having a near-infrared wavelength was used to project ahologram of a sphere into the center of the hollow tube of themicrovasculature structure. The microvasculature structure was suspendedwithin a medium comprising collagen methacrylate, PEGDA, and Eosin Y.FIG. 55F shows the sphere (outlined by the dashed circle) was depositedwithin the lumen of the microvasculature structure without disruptingit. FIG. 55E illustrates the image shown in FIG. 55F. The sphere washolographically printed in its entirety in about 5 milliseconds (ms) atmost.

Example 12—Holographic Printing of a 3D Microvasculature Bed

FIGS. 56A-56B show images of a polymeric microvasculature bed printedusing the methods and systems provided herein. FIG. 56A shows an imageof the vasculature bed during the holographic printing process. Theilluminated areas correspond to a multi-photon laser beam projecting ahologram of the 3D microvasculature bed onto a medium. The mediumincluded a polymeric precursor and a photoinitiator. FIG. 56B shows abright field image of the 3D microvasculature bed after the holographicprinting process is completed.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. It is not intendedthat the invention be limited by the specific examples provided withinthe specification. While the invention has been described with referenceto the aforementioned specification, the descriptions and illustrationsof the embodiments herein are not meant to be construed in a limitingsense. Numerous variations, changes, and substitutions will now occur tothose skilled in the art without departing from the invention.Furthermore, it shall be understood that all aspects of the inventionare not limited to the specific depictions, configurations or relativeproportions set forth herein which depend upon a variety of conditionsand variables. It should be understood that various alternatives to theembodiments of the invention described herein may be employed inpracticing the invention. It is therefore contemplated that theinvention shall also cover any such alternatives, modifications,variations or equivalents. It is intended that the following claimsdefine the scope of the invention and that methods and structures withinthe scope of these claims and their equivalents be covered thereby.

What is claimed is:
 1. A method for printing a three-dimensional (3D)biological material, comprising: (a) providing a media chambercomprising a medium comprising: (i) a plurality of cells and (ii) one ormore polymer precursors; and (b) using at least one energy beam togenerate a plurality of points patterned as a 3D holographic projectionsimultaneously throughout a volume of said medium in said media chamberin accordance with transformed computer instructions for printing said3D biological material in computer memory, to form at least a portion ofsaid 3D biological material comprising: (i) at least a subset of saidplurality of cells, and (ii) a polymer formed from said one or morepolymer precursors, wherein said 3D holographic projection is generatedwithout use of a digital micromirror device.
 2. The method of claim 1,further comprising prior to (b), generating a point-cloud representationor lines-based representation of said 3D biological material in computermemory, and using said point-cloud representation or lines-basedrepresentation to generate said computer instructions.
 3. The method ofclaim 2, further comprising converting said point-cloud representationor lines-based representation into an image, and using said image togenerate said plurality of points patterned as said 3D holographicprojection.
 4. The method of claim 3, wherein said image isdeconstructed and reconstructed prior to generating said plurality ofpoints patterned as said 3D holographic projection simultaneouslythroughout said volume.
 5. The method of claim 2, wherein saidpoint-cloud representation or said lines-based representation comprisesmulti-dimensional structural elements corresponding to said 3Dbiological material.
 6. The method of claim 1, wherein said at least oneenergy beam comprises coherent light.
 7. The method of claim 1, whereinsaid at least one energy beam is phase modulated.
 8. The method of claim1, wherein said one or more polymer precursors comprise at least twodifferent polymeric precursors.
 9. The method of claim 1, furthercomprising repeating (b) to form at least another portion of said 3Dbiological material.
 10. The method of claim 9, wherein said at leastanother portion of said 3D biological material is linked to said 3Dbiological material formed in (b).
 11. The method of claim 9, whereinsaid at least another portion of said 3D biological material is notlinked to said 3D biological material formed in (b).
 12. The method ofclaim 1, wherein (b) further comprises using at least two energy beamsto permit multiple portions of said medium in said media chamber tosimultaneously form said at least said portion of said 3D biologicalmaterial.
 13. The method of claim 12, wherein said at least two energybeams are of identical wavelengths.
 14. The method of claim 12, whereinsaid at least two energy beams are of different wavelengths.
 15. Themethod of claim 1, wherein said at least said portion of said 3Dbiological material comprises microvasculature for providing one or morenutrients to said plurality of cells.
 16. The method of claim 15,wherein said microvasculature has a cross-section from about 1 μm toabout 20 μm.
 17. The method of claim 1, wherein said 3D biologicalmaterial has a thickness or diameter from about 100 μm to about 5 cm.18. The method of claim 1, wherein said medium further comprises aplurality of beads, and wherein in (b) said at least said portion ofsaid 3D biological material, as formed, includes said plurality ofbeads.
 19. The method of claim 18, wherein said plurality of beadsfurther comprise signaling molecules or proteins.
 20. The method ofclaim 19, wherein said signaling molecules or proteins promote formationof said 3D biological material to permit organ function.
 21. The methodof claim 1, wherein said 3D biological material comprises a plurality ofcell-containing scaffolds.
 22. The method of claim 21, wherein a firstcell-containing scaffold of said plurality of cell-containing scaffoldsis coupled to a second cell-containing scaffold of said plurality ofcell-containing scaffolds.
 23. The method of claim 21, wherein saidplurality of cell-containing scaffolds comprise a network, and whereinsaid network comprises a plurality of strands.
 24. The method of claim23, wherein individual strands of said plurality of strands have athickness from about 0.1 nm to about 5 cm.
 25. The method of claim 1,wherein said at least said subset of said plurality of cells comprisescells of at least two different types.
 26. The method of claim 25,wherein in (a), said plurality of cells does not include said cells ofsaid at least two different types, and wherein upon forming said atleast said portion of said 3D biological material, at least a portion ofsaid at least said subset of said plurality of cells is subjected todifferentiation to form said cells of said at least two different types.27. The method of claim 1, wherein said at least one energy beam is amulti-photon energy beam.
 28. The method of claim 1, wherein said mediumfurther comprises glutathione.
 29. The method of claim 27, wherein saidmulti-photon energy beam comprises a two-photon energy beam.
 30. Themethod of claim 1, wherein said 3D holographic projection is generatedusing a spatial light modulator (SLM) that is not a digital micromirrordevice.